Constrained conditionally activated binding proteins

ABSTRACT

The invention relates to COnditional Bispecific Redirected Activation constructs, or COBRAs, that are administered in an active pro-drug format. Upon exposure to tumor proteases, the constructs are cleaved and activated, such that they can bind both tumor target antigens (TTAs) as well as CD3, thus recruiting T cells expressing CD3 to the tumor, resulting in treatment.

I. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application 62/555,943 filed Sep. 8, 2017, U.S.Provisional Patent Application 62/586,627 filed Nov. 15, 2017, and U.S.Provisional Patent Application 62/587,318 filed Nov. 16, 2017, all ofwhich are expressly incorporated herein by reference in their entirety.

II. REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

The sequence listing contained in the file named “118459-5005_ST25.txt”and having a size of 984 kilobytes, has been submitted electronicallyherewith via EFS-Web, and the contents of the txt file are herebyincorporated by reference in their entirety.

III. BACKGROUND OF THE INVENTION

The selective destruction of an individual cell or a specific cell typeis often desirable in a variety of clinical settings. For example, it isa primary goal of cancer therapy to specifically destroy tumor cells,while leaving healthy cells and tissues as intact and undamaged aspossible. One such method is by inducing an immune response against thetumor, to make immune effector cells such as natural killer (NK) cellsor cytotoxic T lymphocytes (CTLs) attack and destroy tumor cells.

The use of intact monoclonal antibodies (mAb), which provide superiorbinding specificity and affinity for a tumor-associated antigen, havebeen successfully applied in the area of cancer treatment and diagnosis.However, the large size of intact mAbs, their poor bio-distribution, lowpotency and long persistence in the blood pool have limited theirclinical applications. For example, intact antibodies can exhibitspecific accumulation within the tumor area. In biodistribution studies,an inhomogeneous antibody distribution with primary accumulation in theperipheral regions is noted when precisely investigating the tumor. Dueto tumor necrosis, inhomogeneous antigen distribution and increasedinterstitial tissue pressure, it is not possible to reach centralportions of the tumor with intact antibody constructs. In contrast,smaller antibody fragments show rapid tumor localization, penetratedeeper into the tumor, and also, are removed relatively rapidly from thebloodstream.

However, many antibodies, including scFvs and other constructs, show “ontarget/off tumor” effects, wherein the molecule is active on non-tumorcells, causing side effects, some of which can be toxic. The presentinvention is related to novel constructs that are selectively activatedin the presence of tumor proteases.

IV. SUMMARY OF THE INVENTION

The present invention provides a number of different proteincompositions for the treatment of cancer. Accordingly, in one aspect,the invention provides “Format 2” proteins comprising, from N- toC-terminal: a first single domain antigen binding domain (sdABD) thatbinds to a human tumor target antigen (TTA) (sdABD-TTA); b) a firstdomain linker; c) a constrained Fv domain comprising: i) a firstvariable heavy domain comprising a vhCDR1, vhCDR2 and vhCDR3; ii) aconstrained non-cleavable linker (CNCL); and iii) a first variable lightdomain comprising vlCDR1, vlCDR2 and vlCDR3; d) a second domain linker;e) a second sdABD-TTA; f) a cleavable linker (CL); g) a constrainedpseudo Fv domain comprising: i) a first pseudo light variable domain;ii) a non-cleavable linker (NCL); and iii) a first pseudo heavy variabledomain; h) a third domain linker; and i) a third sdABD that binds tohuman serum albumin; wherein said first variable heavy domain and saidfirst variable light domain are capable of binding human CD3 but saidconstrained Fv domain does not bind CD3; said first variable heavydomain and said first pseudo variable light domain intramolecularlyassociate to form an inactive Fv; and said first variable light domainand said first pseudo variable heavy domain intramolecularly associateto form an inactive Fv.

In a further aspect, the invention provides “Format 1” proteinscomprising, from N- to C-terminal: a) a first sdABD-TTA; b) a firstdomain linker; c) a constrained Fv domain comprising: i) a firstvariable heavy domain comprising a vhCDR1, vhCDR2 and vhCDR3; ii) aconstrained cleavable linker (CCL); and iii) a first variable lightdomain comprising vlCDR1, vlCDR2 and vlCDR3; d) a second domain linker;e) a second sdABD-TTA; f) a cleavable linker (CL); g) a constrainedpseudo Fv domain comprising: i) a first pseudo light variable domain;ii) a non-cleavable linker (NCL); and iii) a first pseudo heavy variabledomain; h) a third domain linker; and i) a third sdABD that binds tohuman serum albumin; wherein said first variable heavy domain and saidfirst variable light domain are capable of binding human CD3 but saidconstrained Fv domain does not bind CD3; wherein said first variableheavy domain and said first pseudo variable light domainintramolecularly associate to form an inactive Fv; and wherein saidfirst variable light domain and said first pseudo variable heavy domainintramolecularly associate to form an inactive Fv.

In an additional aspect, the invention provides “Format 4” proteinscomprising, from N- to C-terminal: a) a single domain antigen bindingdomain (sdABD) that binds to a human tumor target antigen (TTA)(sdABD-TTA); b) a first domain linker; c) a constrained Fv domaincomprising: i) a first variable heavy domain comprising a vhCDR1, vhCDR2and vhCDR3; ii) a constrained non-cleavable linker (CNCL); and iii) afirst variable light domain comprising vlCDR1, vlCDR2 and vlCDR3; d) acleavable linker (CL); e) a second sdABD that binds to human serumalbumin; f) a domain linker; g) a constrained pseudo Fv domaincomprising: i) a first pseudo light variable domain; ii) a non-cleavablelinker (NCL); and iii) a first pseudo heavy variable domain; whereinsaid first variable heavy domain and said first variable light domainare capable of binding human CD3 but said constrained Fv domain does notbind CD3; wherein said first variable heavy domain and said first pseudovariable light domain intramolecularly associate to form an inactive Fv;and wherein said first variable light domain and said first pseudovariable heavy domain intramolecularly associate to form an inactive Fv.

In a further aspect to the Format 1, Format 2 and Format 4 proteinslisted above, said first variable heavy domain is N-terminal to saidfirst variable light domain and said pseudo light variable domain isN-terminal to said pseudo variable heavy domain.

In a further aspect to the Format 1, Format 2 and Format 4 proteinslisted above, said first variable heavy domain is N-terminal to saidfirst variable light domain and said pseudo variable heavy domain isN-terminal to said pseudo variable light domain.

In a further aspect to the Format 1, Format 2 and Format 4 proteinslisted above, said first variable light domain is N-terminal to saidfirst variable heavy domain and said pseudo light variable domain isN-terminal to said pseudo variable heavy domain.

In a further aspect to the Format 1, Format 2 and Format 4 proteinslisted above, said first variable light domain is N-terminal to saidfirst variable heavy domain and said pseudo variable heavy domain isN-terminal to said pseudo variable light domain.

In an additional aspect, the invention provides Format 1 and 2 proteinswherein said first and second TTA are the same.

In a further aspect, the invention provides Format 1 and 2 proteinswherein said first and second TTA are different.

In an additional aspect, the invention provides Format 1, 2 and 4proteins wherein said first and second TTA are selected from EGFR,EpCAM, FOLR1 and B7H3. These sequences can be selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29; SEQ ID NO:33; SEQ IDNO:37 and SEQ ID NO:41.

In a further aspect, the invention provides Format 1, 2 and 4 proteinswherein said half-life extension domain has SEQ ID NO:45.

In an additional aspect, the invention provides Format 1, 2 and 4proteins wherein said cleavable linker is cleaved by a human proteaseselected from the group consisting of MMP2, MMP9, Meprin A, Meprin B,Cathepsin S, Cathepsin K, Cathespin L, GranzymeB, uPA, Kallekriein7,matriptase and thrombin.

In a further aspect, the invention provides a protein selected from thegroup consisting of Pro186, Pro225, Pro226, Pro233, Pro311, Pro312,Pro313, Pro495, Pro246, Pro254, Pro255, Pro256, Pro420, Pro421, Pro432,Pro479, Pro480, Pro187, Pro221, Pro222, Pro223, Pro224, Pro393, Pro394,Pro395, Pro396, Pro429, Pro430 and Pro431.

In an additional aspect, the invention provides nucleic acids encoding aFormat 1, Format 2 or Format 4 protein as described herein, as well asexpression vectors and host cells comprising the nucleic acids encodingthe protein.

In a further aspect, the invention provides methods of making theproteins of the invention and methods of treating patients in needthereof.

In an additional aspect, the invention provides compositions comprising“Format 3A” pairs of pro-drug proteins, comprising: a) a first proteincomprising, from N- to C-terminal: i) a first sdABD-TTA; ii) a firstdomain linker; iii) a pseudo Fv domain comprising, from N- toC-terminal: 1) a variable heavy chain comprising a vhCDR1, vhCDR2 andvhCDR3; 2) a cleavable linker; and 3) a first pseudo variable lightdomain comprising iVLCDR1, iVLCDR2 and iVLCDR3; iv) a second domainlinker; v) a sdABD-HSA; a) a first second protein comprising, from N- toC-terminal: i) a third sdABD that binds to a human tumor target antigen;ii) a third domain linker; iii) a pseudo Fv domain comprising, from N-to C-terminal: 1) a variable light chain comprising a VLCDR1, VLCDR2 andVLCDR3; 2) a cleavable linker; and 3) a first pseudo variable heavydomain comprising iVHCDR1, iVHCDR2 and iVHCDR3; iv) a fourth domainlinker; v) a sdABD-HSA; wherein said first variable heavy domain andsaid first variable light domain are capable of binding human CD3 whenassociated; wherein said first variable heavy domain and said firstpseudo variable light domain intermolecularly associate to form aninactive Fv; wherein said first variable light domain and said firstpseudo variable heavy domain intermolecularly associate to form aninactive Fv; and wherein said first and third sdABD are selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ IDNO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29; SEQ IDNO:33; SEQ ID NO:37 and SEQ ID NO:41.

In a further aspect, the invention provides compositions comprising“Format 3B” pairs of pro-drug proteins, comprising a) a first proteincomprising, from N- to C-terminal: i) a first sdABD-TTA; ii) a firstdomain linker; iii) a second sdABD-TTA; iv) a second domain linker; iii)a pseudo Fv domain comprising, from N- to C-terminal: 1) a variableheavy chain comprising a vhCDR1, vhCDR2 and vhCDR3; 2) a cleavablelinker; and 3) a first pseudo variable light domain comprising iVLCDR1,iVLCDR2 and iVLCDR3; iv) a third domain linker; and v) a sdABD-HSA; a) afirst second protein comprising, from N- to C-terminal: i) a thirdsdABD-TTA; ii) a fourth domain linker; iii) a fourth sdABD-TTA; iv) afifth domain linker; iii) a pseudo Fv domain comprising, from N- toC-terminal: 1) a variable light chain comprising a VLCDR1, VLCDR2 andVLCDR3; 2) a cleavable linker; and 3) a first pseudo variable heavydomain comprising iVHCDR1, iVHCDR2 and iVHCDR3; iv) a sixth domainlinker; v) a sdABD-HSA; wherein said first variable heavy domain andsaid first variable light domain are capable of binding human CD3 whenassociated; wherein said first variable heavy domain and said firstpseudo variable light domain intermolecularly associate to form aninactive Fv; and wherein said first variable light domain and said firstpseudo variable heavy domain intermolecularly associate to form aninactive Fv.

In an additional aspect, Format 3A and Format 3B proteins have sdABD-HSAthat have SEQ ID NO:45.

In a further aspect, Format 3A and Format 3B proteins have sdABD-TTAthat binds to a TTA selected from EGFR, EpCAM, FOLR1 and B7H3. ThesdABD-TTAs can be selected from the group consisting of SEQ ID NO:1, SEQID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ IDNO:25, SEQ ID NO:29; SEQ ID NO:33; SEQ ID NO:37 and SEQ ID NO:41.

In a further aspect, the invention provides nucleic acid compositionscomprising first nucleic acids that encode the first protein members ofthe prodrug pair and second nucleic acids that encode the second proteinmembers of the pairs, and expression vectors and host cells containingthe nucleic acids.

V. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the “format 1” type of protease activation of the presentinvention, referred to herein as “constrained, cleavable constructs” or“cc constructs”. In this embodiment, a representative construct isPro140: there are ABDs for two TTA (as depicted in FIG. 1, these areboth the same, although as described herein they can be different). Uponcleavage, the prodrug construct splits into three components, onecontaining an α-TTA domain linked via a domain linker to an active VH ofαCD3, the second containing an α-TTA domain linked via a domain linkerto an active VL of αCD3, and a “leftover” piece comprising the half lifeextension domain linked to the inactive VH and VL. The two activevariable domains then are free to associate to form a functionalanti-CD3 binding domain. It should be noted that in “format 1”embodiments, the resulting active component is trivalent: there ismonovalent binding to CD3 and bivalent binding to the TTA, rendering abispecific binding protein, although in some cases this trivalency couldbe trispecifics, with monovalent binding to CD3, monovalent binding to afirst TTA and monovalent binding to a second TTA. FIG. 1 also shows ananti-human serum albumin (HSA) domain as a half-life extension domain,in many embodiments an sdABD as defined herein, although as discussedherein, this is optional and/or can be replaced by other half-lifeextension domains; additionally, the half-life extension domain can alsobe N-terminal to the construct or internal as well. FIG. 1 also has theVH and VL of the Fv and iVH and iVL of the pseudo Fv in a specificorder, e.g. from N- to C-terminal, VH-linker-VL (and iVL-linker-iVH)although as will be appreciated by those in the art, these can bereversed (VL-linker-VH and iVH-linker-iVL). Alternatively, one of theseFvs can be in one orientation and the other in the other orientation,although the expression of protein in the orientation as shown here wassurprisingly higher than the other orientations.

FIG. 2 depicts the “format 2” type of protease activation of the presentinvention, referred to herein as “constrained, non-cleavableconstructs”, or “CNCL constructs”, also sometimes referred to herein as“dimerization constructs” as discussed herein. These constructs do notisomerize as discussed herein. Upon cleavage, two prodrug constructsplits into four components, two half-life extension domains (in thiscase, sdABDs to HSA) linked to two pseudo domains (which may or may notbe able to self-associate, depending on the length of the linkers andthe inactivating mutations), and two active moieties that self-assembleinto a dimeric active moiety that contains four anti-TTA domains (whichcan be all the same or two are the same and the other two aredifferent). It should be noted that in “format 2” embodiments, theresulting active component is hexavalent: there is bivalent binding toCD3 and quadrivalent binding to the TTA, rendering a bispecific bindingprotein, although in some cases this hexavalency could be trispecifics,with bivalent binding to CD3, bivalent binding to a first TTA andbivalent binding to a second TTA. FIG. 2 also shows an anti-human serumalbumin (HSA) domain as a half-life extension domain, in manyembodiments an sdABD as defined herein, although as discussed herein,this is optional and/or can be replaced by other half-life extensiondomains; additionally, the half-life extension domain can also beN-terminal to the construct or internal as well. FIG. 2 also has the VHand VL of the Fv and iVH and iVL of the pseudo Fv in a specific order,e.g. from N- to C-terminal, VH-linker-VL (and iVL-linker-iVH) althoughas will be appreciated by those in the art, these can be reversed(VL-linker-VH and iVH-linker-iVL). Alternatively, one of these Fvs canbe in one orientation and the other in the other orientation, althoughthe expression of protein in the orientation as shown here wassurprisingly higher than the other orientations.

FIG. 3A-FIG. 3B depict “format 3” type of constructs, also sometimesreferred to as “hemi-constructs” or “hemi-COBRA™” as outlined herein, asthese are two different polypeptide chains that together make up an MCEtherapeutic as is further discussed herein. In this embodiment, theconstructs are delivered in pairs, with the pre-cleavage intramolecularself-assembly resulting in inactive anti-CD3 Fv domains. Upon cleavage,the inert variable domains are released, and the two active variabledomains then intermolecularly assemble, to form an active anti-CD3binding domain. The two sdABD-TTAs bind to the corresponding receptor onthe tumor cell surface, and the cleavage is done by a protease. Thisallows the intermolecular assembly, since the molecules are physicallyheld in place, favoring the assembly of the active anti-CD3 domain. Asabove for formats 1 and 2, in this embodiment, the N- to C-terminalorder of the variable domains can be reversed, or mixed as well.Furthermore, the sdABD(HSA) can be either at the N- or C-terminus ofeach hemi-construct. Pro16 has the sdABD(HSA) at the C terminus andPro17 has it at the N-terminus (see Pro19, SEQ ID NO:XX, has thesdABD(HSA) at the C-terminus). FIG. 3A shows Format 3 constructs with asingle sdABD-TTA domain per hemi-construct, and FIG. 3B shows Format 3constructs with two sdABD-TTAs per hemi-construct, in a “dual targeting”or “hetero-targeting” format. Note that FIG. 3B uses FOLR1 and EGFR asthe two TTAs, but other combinations as outlined herein can also beused.

FIG. 4 depicts “format 4” type of constructs that are similar to “format2” constructs but have only a single sdABD-TTA. The figure shows thesdABD-TTA to EGFR, but as will be appreciated by those in the art, otherTTA can be used as well. Upon cleavage, the prodrug construct splitsinto two components, a half-life extension domain (in this case, sdABDsto HSA) linked to a pseudo Fv and an active moiety, that in the presenceof a second active moiety from a different cleaved molecule,self-assembles into a dimeric active moiety that contains two anti-TTAdomains. It should be noted that in “format 4” embodiments, theresulting active component is quadrivalent: there is bivalent binding toCD3 and bivalent binding to the TTA, rendering a bispecific bindingprotein. FIG. 4 also shows an anti-human serum albumin (HSA) domain as ahalf-life extension domain, in many embodiments an sdABD(½) as definedherein, although as discussed herein, this is optional and/or can bereplaced by other half-life extension domains; additionally, thehalf-life extension domain can also be N-terminal to the construct orinternal as well. FIG. 4 also has the VH and VL of the Fv and iVH andiVL of the pseudo Fv in a specific order, e.g. from N- to C-terminal,VH-linker-VL (and iVL-linker-iVH) although as will be appreciated bythose in the art, these can be reversed (VL-linker-VH andiVH-linker-iVL). Alternatively, one of these Fvs can be in oneorientation and the other in the other orientation, although theexpression of protein in the orientation as shown here was surprisinglyhigher than the other orientations.

FIG. 5A-FIG. 5G depict a number of sequences of the invention. Forantigen binding domains, the CDRs are underlined. As is more fullyoutlined herein, these domains can be assembled in a wide variety ofconfigurations in the present invention, including “format 1”, “format2”, “format 3” and “format 4” orientations. Of note is that SEQ ID NO:90is cleaved by MMP9 slightly faster than SEQ ID NO:s 75 and 76, and SEQID NO:91 is cleaved slower than SEQ ID NO:s 75 and 76.

FIG. 6A-FIG. 6B depict a number of suitable protease cleavage sites. Aswill be appreciated by those in the art, these cleavage sites can beused as cleavable linkers. In some embodiments, for example when moreflexible cleavable linkers are required, there can be additional aminoacids (generally glycines and serines) that are either or both N- andC-terminal to these cleavage sites.

FIG. 7A-FIG. 7D depict some data associated with the “Format 3” or“hemi-COBRA™” structures. This shows that Format 3 constructs bindco-operatively to CD3 after cleavage by protease (in this case EKprotease, although any of the protease cleavage sites outlined hereinand depicted in FIG. 5 and FIG. 6 can be used) and create a CD3 bindingsite, as shown by a sandwich FACS analysis.

FIG. 8A-FIG. 8D shows that the protease cleavage co-operativelyactivates T-cell killing of EGFR+ target cells with complimentaryhemi-COBRA™ pairs. FIG. 8A and FIG. 8B show that the constructs inisolation, but cleaved with different concentrations of protease, do notaffect target cell viability. However, FIG. 8C shows that incombination, in the presence of protease, target cell viability issignificantly diminished. FIG. 8D shows the general mechanism.

FIG. 9 shows some non-target controls for use in the assays to testefficacy of the Format 1 constructs.

FIG. 10A-FIG. 10F shows the generation of an active CD3 binding domainis dependent on target binding of both “arms”, e.g. the sdABD-TTAdomains, one of which is on each of the two constructs. The TDCC assaywas done as described in the Examples.

FIG. 11 shows the schematic of suitable hemi-COBRA™ pairs. “Mep” standsfor a meprin protease cleavage site, “His-6” is a tag as more fullydiscussed herein, ST14 is a matriptase protease cleavage site and “Thb”is a thrombin protease cleavage site.

FIG. 12A-FIG. 12C shows the TDCC data associated with the constructs ofFIG. 11. FIG. 12A shows that addition of pre-cleaved hemi-COBRA pairsresults in efficacy on OvCAR8 cells, FIG. 12B shows that addition ofpre-cleaved hemi-COBRA pairs results in efficacy on HCT116 cells, andFIG. 12C shows that addition of pre-cleaved hemi-COBRA pairs results inefficacy on LoVo cells, all of which are cancer cell lines.

FIG. 13A-FIG. 13B shows that the MMP9 linker is stable in vivo. NSG micewere administered a single intravenous bolus dose of either Pro40 (MMP9cleavable), Pro74 (non-cleavable) via the tail vein at a dose level of0.5 mg/kg. The dose solution for each compound was prepared in a vehicleof 25 mM citric acid, 75-mM L-arginine, 75 mM NaCl and 4% sucrose pH7.0. Two blood samples were collected at preselected times from eachanimal, one towards the beginning of the study, collected by orbitalbleed or submandibular bleed, and another at the terminal time point bycardiac puncture. The time points for blood collection were 0.083, 1, 6,24, 72, and 168 h. Plasma was prepared from each individual blood sampleusing K₂ EDTA tubes. Concentrations were determined using an MSD assaywith a MAb specific to the anti-HSA sdABD and detected with the EGFRextracellular domain.

FIG. 14 depicts the schematics of the Format 3A hemi-COBRA™ constructsused in the experiments depicted in FIG. 15. Pro51 is the positivecontrol, as it is “always on”, since it forms an active anti-CD3 Fv.Pro98 is a negative control, since it's sdABD is directed against henegg lysozyme, which isn't expressed by the tumor. Pro77 and Pro53 arethe pro-drug Format 3A pair, using sdABDs against EGFR and an MMP9cleavage site. Pro74 and Pro72 is a negative control Format 3A pair,since they don't have cleavage sites.

FIG. 15 shows that Format 1 constructs work to regress tumors in vivo,using two different tumor cell lines implanted into mice using theprotocols in the Examples. Anti-tumor activity with the hemi-COBRAconstructs (Pro77 and Pro53) was dependent on the inclusion of both theanti-EGFR sdABDs and the MMP9 cleavable linkers, along with the activeanti-CD3 Fv.

FIG. 16 shows the schematic of the next generation format, a full lengthconstruct that has two pseudo Fv domains with cleavable sites betweenthem, as is generally described in US 2018/0134789, hereby incorporatedby reference. However, as shown in the following figures, this firstgeneration full length construct does not show very good conditionality,as it can isomerize to form both an active and inactive construct.

FIG. 17 shows that the Format 3A construct pairs actually show betterconditionality than the Pro100 first generation full length constructs.

FIG. 18 depicts additional first generation full length constructs thatwere tested in Figure.

FIG. 19 shows that the first generation constructs show high activityeven in the uncleaved format, e.g. poor conditionality.

FIG. 20 shows that the first generation full length constructs show twomonomer peaks on analytical SEC.

FIG. 21 shows the schematic of the reason for the noncleaved activity,which is that the full length first generation constructs isomerize toform two conformations, one which is inactive since there is no activeanti-CD3 Fv formed (the “bivalent scFv”), and the other which is activein the absence of protease, a “single chain diabody” type ofconfiguration. See PEDS 23(8):667-677 (2010).

FIG. 22 shows the results of a TDCC assay, run at 37 C for 2 days, withthe first generation single chain constructs. The results show that theuncleaved constructs show strong killing. These results led to thegeneration of the Format 1 constructs.

FIG. 23A-FIG. 23G shows Format 1 constructs used in the invention. Aswill be appreciated by those in the art and described herein, these aredepicted with a sdABD-EGFR targeting moiety, although sdABDs to otherTTAs can be used.

FIG. 24 shows that the Format 1 constructs (Pro140 in this case) form asingle isomer that is stable at 37 C.

FIG. 25 depicts that Format 1 constructs have very low binding to humanCD3 in the uncleaved format, as measured by an Octet assay. The top lineis Pro120, the middle line is Pro51 (the positive control) and thebottom lines are Pro140 held at either 4 C or 37 C for 3 days.

FIG. 26 similarly depicts that the Format 1 constructs have very lowTDCC activity in the uncleaved form.

FIG. 27 depicts a specific Format 1 construct, Pro140, used in in vivotesting, using sdABD-EGFR as the targeting moieties and an MMP9 cleavagesite.

FIG. 28A-FIG. 28B shows tumor regression using a Format 1 construct.

FIG. 29 depicts that due to the cleavage site in the constrained Fv,several different fragments can be generated: a partially cleavedfragment and the fully cleaved fragment. Surprisingly, the partiallycleaved format is more active than the fully cleaved format, leading tothe generation of Format 2.

FIG. 30 shows a number of Format 2 schematics, all of which usesdABD-EGFR targeting domains, although as outlined herein and listed inthe sequences, sdABDs to other TTAs can be used. Pro51 and Pro201 arepositive controls (in an active “hemi” configuration), and Pro214 is afull length negative control, as there is no cleavage site.

FIG. 31 shows the TDCC activity of Format 2 construct Pro187, which usesa meprin cleavage site. Pro187 in the TDCC assay was 1200-fold moreactive when added pre-cleaved than when added uncleaved. The pre-cleavedPro187 demonstrated activity that fell between the positive controlsPro51 and Pro201. The uncleaved Pro187 demonstrated activity similar toPro214, which does not contain a protease cleavable linker.

FIG. 32 shows the TDCC activity of Format 2 construct Pro186, which usesn MMP9 cleavage site. Pro186 in the TDCC assay was 18-fold more activewhen added pre-cleaved than when added uncleaved. The pre-cleaved Pro186demonstrated activity that fell between the positive controls Pro51 andPro201. The uncleaved Pro186 demonstrated more activity than Pro214,which does not contain a protease cleavable linker.

FIG. 33 depicts that the Pro186 construct binds to cells that havedifferent levels of EGFR receptors, with CHO cells not expressing EGFRon the cell surface. Pro186 saturates cells expressing differing levelsof EGFR at similar COBRA concentrations.

FIG. 34 shows the schematics of Format 2 constructs used in the in vivostudies of Figure, all of which use sdABD-EGFR targeting domains.

FIG. 35 shows that the Format 2 construct Pro186 is highly efficaciousat both concentrations, and better than the Format 1 construct Pro140 atthe lower concentration.

FIG. 36 depicts a number of Format 2 constructs based on Pro186 but withdifferent protease cleavage sites. While all of these constructs utilizesdABD-EGFRs for both targeting domains, other sdABDs to different TTAscan be used, and can be the same or different. That is, bothhomo-targeting (both targeting sdABDs to the same TTA) orhetero-targeting (one sdABD to a first TTA and the other to a differentTTA) can be done.

FIG. 37 depicts the schematics for different Format 2 constructs thatvary linker length between the Fv domains. These are shown using an MMP9cleavage site, although others can be used as outlined herein.Similarly, while all of these constructs utilize sdABD-EGFRs for bothtargeting domains, other sdABDs to different TTAs can be used, and canbe the same or different.

FIG. 38 shows that the linker length for the pseudo Fv can be varied,e.g. that a Format 2 construct with a short linker between the active Fv(“short active”) with a longer linker between the pseudo Fv (“longinactive”) exhibits similar activity to a “short active” with a “shortinactive”. Thus conditionality of the COBRA construct is not dependenton both the active and inactive scFv linkers being constrained; as longas one of them is constrained, single chain diabody folding appears tobe favored over bivalent scFv folding.

FIG. 39 shows that the linker length for the active Fv can be varied,e.g. that Format 2 constructs with “long active” and “short inactive”behaves similarly to a “short active” and “short inactive” construct.Thus conditionality of the COBRA construct is not dependent on both theactive and inactive scFv linkers being constrained; as long as one ofthem is constrained, single chain diabody folding appears to be favoredover bivalent scFv folding.

FIG. 40A-FIG. 40C shows the schematics for a number of differentconstructs. Pro188 is a Format 1 construct which is similar to Pro140except with a long linker (16mer) in the pseudo Fv. Pro189 and Pro190(Format 2 constructs) are similar to Pro186 and Pro187 except with along linker (16mer) in the pseudo Fv domain. Pro191 and Pro192 (alsoFormat 2 constructs) are similar to Pro189 and Pro190 except they havean additional cleavage site upstream of the sdABD(½). Pro193 (Format 4)has a single EGFR targeting domain, the iVH and iVL rearranged to be inreversed order, and an additional cleavage site upstream of thesdABD(½). Pro195 is a Format 2 construct similar to Pro186, withtargeting domains that bind to the same TTA, EGFR, but to differentepitopes. Pro196, Pro197 and Pro198 are Format 2 constructs withrearranged variable domains.

FIG. 41 depicts the fact that different sdABD clones directed to humanFOLR1 show differential killing. A Pro22 type construct (Pro51 with aFLAG sequence instead of a NCL) that binds to human FOLR1 was comparedto a Pro22-EGFR construct against a number of cell line families.

FIG. 42 depicts the schematics for four sdABD-FOLR1 constructs,including the use of the Pro201 positive control using sdABD-EGFR2 (withtwo molecules intermolecularly associating to form two active Fvsagainst CD3), and two Format 2 test articles, Pro311, using the h77.2sdABD and Pro312 using the h59.3 sdABD, as well as two negativecontrols, Pro299, using the h77.2 sdABD and Pro303 using the h59.3sdABD.

FIG. 43 depicts the schematics of the Format 2 constructs for theFOLR/MMP9 in vivo design.

FIG. 44 shows the efficacy of the Pro312 construct in vivo, anddemonstrates the MMP9 cleavable linker is necessary for anti-tumoractivity.

FIG. 45 depicts the schematics of some formats using sdABDs to humanB7H3 (sdABD-B7H3), including Pro244, the positive control (usingsdABD-B7H3 (hF7) (with two molecules intermolecularly associating toform two active Fvs against CD3), and two Format 2 test articles,Pro225, a Format 2 construct, and Pro295, the negative control lacking acleavage site.

FIG. 46 shows that Pro225 has great conditionality as compared to thecontrol, Pro295.

FIG. 47 shows that a Format 2 construct of using a meprin linker,Pro373, shows great conditionality compared to Pro295.

FIG. 48 depicts a number of sdABD-B7H3 (using the hF12 sequence)constructs, showing the Pro51 positive control using sdABD-EGFR, thePro244 positive control using sdABD-hF12 B7H3, the test construct,Pro226, and the negative control Pro296 without a cleavage site.

FIG. 49 shows the good conditionality of the Pro226 construct in a TDCCassay.

FIG. 50 shows the humanization of sdABDs to human EpCAM.

FIG. 51 shows the schematics of a number of Formats: Pro22hVIB13 andPro205 are positive controls, Pro199 is a Format 2 construct and Pro175is the negative control.

FIG. 52 shows the TDCC activity of sdABD-EpCAM constructs, showing goodconditionality.

FIG. 53A-FIG. 53B shows the TDCC activity of sdABD-EpCAM Pro199construct, showing good conditionality in HT29 and LoVo cell models.

FIG. 54A-FIG. 54B shows the TDCC activity of sdABD-EpCAM Pro200construct, showing good conditionality in HT29 and LoVo cell models.

FIG. 55 shows a schematic of Pro255, which uses two different sdABD-TTA,one to EGFR (sdABD-EGFR) and the other to EpCAM (sdABD-EpCAM), ascompared to Pro199, with dual EpCAM sdABDs. These are sometimes referredto herein as “hetero-targeting” constructs, in this case, a Format 2construct.

FIG. 56 shows that the Pro255 dual targeting molecule with an MMP9cleavage site, shows good conditionality.

FIG. 57A-FIG. 57D shows the results of experiments on three differentcell types. First, Raji transfectants were created with similarexpression levels of EpCAM, EGFR and EpCAM+ EGFR (data not shown). ThenPro255, which targets both EpCAM and EGFR, was tested in TDCC assaysusing each cell type. FIG. 57A shows the parental Raji line, thatdoesn't express either receptor. FIG. 57B shows conditionality on theEpCAM line. FIG. 57C shows conditionality on the EGRF line. FIG. 57Dshows conditionality on the EpCAM/EGFR line.

FIG. 58 depicts the schematics of a Format 4 construct, Pro258.

FIG. 59A-FIG. 59B shows that Pro258 is conditional in both FBS and humanserum. The conditionality of the MMP9 linker is underestimated due tothe MMP9 activity in the culture. Interestingly, Pro51 TDCC activity isinhibited by HSA binding while Pro258 TDCC activity is similar to Pro51in the presence of HSA. Finally, the Pro258 conditionality is somewhatenhanced in the presence of HSA by 6×.

FIG. 60A-FIG. 60C shows the cleavage of the MMP9 substrate by otherMMPs.

FIG. 61A-FIG. 61B shows some of the exemplary constructs and theirformats.

FIG. 62A-FIG. 62U shows a number of sequences of the invention, althoughmany additional sequences are also found in the sequence listing. CDRsare underlined and bolded, linkers are double underlined (with cleavablelinkers being italicized and double underlined) and domain separationsare indicated by “/”. All His6 tags are optional, as they can be used toreduce immunogenicity in humans as well as be purification tags.

VI. DETAILED DESCRIPTION OF THE INVENTION A. Introduction

The present invention is directed to methods of reducing the toxicityand side effects of bispecific antibodies (including antibody-likefunctional proteins) that bind to important physiological targets suchas CD3 and tumor antigens. Many antigen binding proteins, such asantibodies, can have significant off-target side effects, and thus thereis a need to only activate the binding capabilities of a therapeuticmolecule in the vicinity of the disease tissue, to avoid off-targetinteractions. Accordingly, the present invention is directed tomultivalent conditionally effective (“MCE”) proteins that have a numberof functional protein domains. In general, one of these domains is anantigen binding domain (ABD) that will bind a target tumor antigen(TTA), and another is an ABD that will bind a T-cell antigen such as CD3under certain conditions. Additionally, the MCE proteins also includeone or more protease cleavage sites. That is, the therapeutic moleculesare made in a “pro-drug” like format, wherein the CD3 binding domain isinactive until exposed to a tumor environment. The tumor environmentcontains proteases, such that upon exposure to the protease, the prodrugis cleaved and becomes active.

This is generally accomplished herein by using proteins that include a“pseudo” variable heavy domain and a “pseudo” variable light domaindirected to the T-cell antigen such as CD3, that restrain the CD3 Fvs ofthe MCE into an inactive format as is discussed herein. As the TTAtargets the MCE into the proximity of the tumor, the MCE is thus exposedto the protease. Upon cleavage, the active variable heavy domain andactive light domain are now able to pair to form one or more active ABDsto CD3 and thus recruit T cells to the tumor, resulting in treatment.

In general, the CD3 binding domain (“Fv”) is in a constrained format,wherein the linker between the active variable heavy domain and theactive variable light domain that traditionally form an Fv is too shortto allow the two active variable domains to bind each other; this isreferred to as “constrained linker”; these can be constrained andcleavable (CCL, as used in Format 1) or constrained and not cleavable(CNCL, as used in Format 2). Rather, in the prodrug (e.g. uncleaved)format, the prodrug polypeptide also comprises a “pseudo Fv domain”. Thepseudo Fv domain comprises a variable heavy and light domain, withstandard framework regions, but “inert” or “inactive” CDRs. The pseudoFv domain also has a constrained linker between the inactive variableheavy and inactive variable light domains. Since neither Fv nor pseudoFv domains can self-assemble due to the steric constraints, there is anintramolecular assembly that pairs the aVL with the iVH and the aVH withthe iVL, due to the affinity of the framework regions of each. However,due to the “inert” CDRs of the pseudo domain, the resulting ABDs willnot bind CD3, thus preventing off target toxicities. However, in thepresence of proteases that are in or near the tumor, the prodrugconstruct is cleaved such that the pseudo-Fv domain is released from thesurface and thus allows the “real” variable heavy and variable lightdomains to associate intermolecularly (e.g. two cleaved constructs cometogether), thus triggering active CD3 binding and the resulting tumorefficacy. These constructs are generally referred to herein asCOnditional Bispecific Redirected Activation constructs, or “COBRAs™”.The stability of the intramolecular assembly is shown by theconditionality experiments herein, whereby in the absence of protease,the uncleaved constructs have no activity (e.g. no active CD3 bindingdomain is formed).

Interestingly, for ease of description, while these constructs are allreferred to herein as “constrained”, additional work shows that theintramolecular assembly is favored even if one of the Fv domains is notconstrained, e.g. one of the domains can have a longer, flexible linker.That is, as shown in Figure, Figure and Figure, intramolecular assemblystill occurs (e.g. the uncleaved constructs are inactive in the absenceof protease cleavage) if only one of the Fv domains, either the one withan active VL and VH, or the pseudo Fv domain, is constrained. However,in the current systems, when both linkers are constrained, the proteinhas better expression. However, as will be appreciated by those of skillin the art, any of the Format 1, Format 2 or Format 4 constructs hereincan have one of these Fv domains with an “unconstrained” or “flexible”linker. For ease of reference, the constructs are shown with both Fvdomains in a constrained format.

The constructs and formats of the invention are variations overinventions described in WO2017/156178, hereby expressly incorporated byreference in its entirety. As shown in Figure, previous constructs havethe ability to isomerize due to the presence of two sets of VH and VLdomains in a single polypeptide, forming both a bivalent scFv and asingle chain diabody. Even after purification of each isoform, thebivalent construct can still reach equilibrium with the diabody isoform.As the single chain diabody has the ability to bind to CD3 in theabsence of protease cleavage, the utility of the construct isdiminished.

To solve this issue, the present invention provides for four separatetypes of constructs to accomplish this conditional activation. Theprodrug activation can happen in one of four general ways, as isgenerally shown in the Figures. In FIG. 1, a “format 1” mechanism isshown. In this embodiment, the prodrug construct has two cleavage sites:one between the VH and vl domains of the constrained Fv, thus freeingthe two variable domains to associate, and a second at a site thatreleases the pseudo Fv domain from the prodrug construct, leaving twomolecules that associate due to the innate self-assembly of the variableheavy and variable light domains, each having an antigen binding domainto a tumor antigen as well, thus allowing the recruitment of T cells tothe tumor site.

In an alternate embodiment, the prodrug construct is shown in FIG. 2, a“format 2” mechanism. In this embodiment, the domain linker between theactive variable heavy and active light chains is a constrained but notcleavable linker (“CNCL”). In the prodrug format, the inactive VH and VLof the constrained pseudo Fv domain associate with the VH and VL of theconstrained Fv domain, such that there is no CD3 binding. However, oncecleavage in the tumor environment happens, two different activatedproteins, each comprising an active variable heavy and light domain,associate to form two anti-CD3 binding domains.

In addition to the “single chain protein” COBRA formats discussed above,where all of the components are contained on a single amino acidsequence, there are also constructs that rely on two proteins“hemi-COBRAs”, which act in pairs, as shown in FIG. 3. In thisembodiment, each protein has one active and one inert variable domainseparated by a protease cleavage site. Each molecule contains a TTAbinding domain, such that when the molecules are bound to the TTA andexposed to tumor protease, the inert domains are cleaved off and the twoactive variable domains self-assemble to form an anti-CD3 bindingdomain.

Furthermore, the invention provides “format 4” constructs as well, asdepicted in FIG. 4. These are similar to the “format 2” designs, exceptthat a single ABD to a TTA is used, such that upon cleavage, two of thepro-drug molecules now form a tetravalent, bispecific constructcontaining two active anti-CD3 domains, as is further described below.

Accordingly, the formats and constructs of the invention find use in thetreatment of disease.

B. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids or any non-natural analogues thatmay be present at a specific, defined position. In many embodiments,“amino acid” means one of the 20 naturally occurring amino acids. By“protein” herein is meant at least two covalently attached amino acids,which includes proteins, polypeptides, oligopeptides and peptides.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence or an alteration toa moiety chemically linked to a protein. For example, a modification maybe an altered carbohydrate or PEG structure attached to a protein. Forclarity, unless otherwise noted, the amino acid modification is alwaysto an amino acid coded for by DNA, e.g. the 20 amino acids that havecodons in DNA and RNA. The preferred amino acid modification herein is asubstitution.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For clarity, a protein which hasbeen engineered to change the nucleic acid coding sequence but notchange the starting amino acid (for example exchanging CGG (encodingarginine) to CGA (still encoding arginine) to increase host organismexpression levels) is not an “amino acid substitution”; that is, despitethe creation of a new gene encoding the same protein, if the protein hasthe same amino acid at the particular position that it started with, itis not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence.

The polypeptides of the invention specifically bind to CD3 and targettumor antigens (TTAs) such as target cell receptors, as outlined herein.“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁴M, at least about 10⁻⁵ M, at least about10⁻⁶ M, at least about 10⁻⁷M, at least about 10⁻⁸M, at least about 10⁻⁹M, alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, atleast about 10⁻¹² M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction. Binding affinity is generally measured using a Biacoreassay or Octet as is known in the art.

By “parent polypeptide” or “precursor polypeptide” (including Fc parentor precursors) as used herein is meant a polypeptide that issubsequently modified to generate a variant. Said parent polypeptide maybe a naturally occurring polypeptide, or a variant or engineered versionof a naturally occurring polypeptide. Parent polypeptide may refer tothe polypeptide itself, compositions that comprise the parentpolypeptide, or the amino acid sequence that encodes it. Accordingly, by“parent Fc polypeptide” as used herein is meant an unmodified Fcpolypeptide that is modified to generate a variant, and by “parentantibody” as used herein is meant an unmodified antibody that ismodified to generate a variant antibody.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound. A range of suitable exemplary target antigens are describedherein.

By “target cell” as used herein is meant a cell that expresses a targetantigen. Generally, for the purposes of the invention, target cells areeither tumor cells that express TTAs or T cells that express the CD3antigen.

By “Fv” or “Fv domain” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of an antigen bindingdomain, generally from an antibody. Fv domains usually form an “antigenbinding domain” or “ABD” as discussed herein, if they contain active VHand VL domains (although in some cases, an Fv containing a constrainedlinker is used, such that an active ABD isn't formed prior to cleavage).As discussed below, Fv domains can be organized in a number of ways inthe present invention, and can be “active” or “inactive”, such as in ascFv format, a constrained Fv format, a pseudo Fv format, etc. It shouldbe understood that in the present invention, in some cases an Fv domainis made up of a VH and VL domain on a single polypeptide chain, such asshown in FIG. 1 and FIG. 2 but with a constrained linker such that anintramolecular ABD cannot be formed. In these embodiments, it is aftercleavage that two active ABDs are formed. In some cases an Fv domain ismade up of a VH and a VL domain, one of which is inert, such that onlyafter cleavage is an intermolecular ABD formed. As discussed below, Fvdomains can be organized in a number of ways in the present invention,and can be “active” or “inactive”, such as in a scFv format, aconstrained Fv format, a pseudo Fv format, etc. In addition, asdiscussed herein, Fv domains containing VH and VL can be/form ABDs, andother ABDs that do not contain VH and VL domains can be formed usingsdABDs.

By “variable domain” herein is meant the region of an immunoglobulinthat comprises one or more Ig domains substantially encoded by any ofthe Vκ, Vλ, and/or VH genes that make up the kappa, lambda, and heavychain immunoglobulin genetic loci respectively. In some cases, a singlevariable domain, such as a sdFv (also referred to herein as sdABD) canbe used.

In embodiments utilizing both variable heavy (VH) and variable light(VL) domains, each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four “frameworkregions”, or “FRs”, arranged from amino-terminus to carboxy-terminus inthe following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, the VH domainhas the structure vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4 and theVL domain has the structurevlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. As is more fully describedherein, the vhFR regions and the vlFR regions self assemble to form Fvdomains. In general, in the prodrug formats of the invention, there are“constrained Fv domains” wherein the VH and VL domains cannot selfassociate, and “pseudo Fv domains” for which the CDRs do not formantigen binding domains when self associated.

The hypervariable regions confer antigen binding specificity andgenerally encompasses amino acid residues from about amino acid residues24-34 (LCDR1; “L” denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3)in the light chain variable region and around about 31-35B (HCDR1; “H”denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavychain variable region; Kabat et al., SEQUENCES OF PROTEINS OFIMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and/or those residues forminga hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52 (LCDR2) and91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1),53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region;Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs of theinvention are described below.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated (inherent) CDRs. Accordingly, the disclosure of eachvariable heavy region is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2and vhCDR3) and the disclosure of each variable light region is adisclosure of the vlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3).

A useful comparison of CDR numbering is as below, see Lafranc et al.,Dev. Comp. Immunol. 27(1):55-77 (2003):

TABLE 1 Kabat + Chothia IMGT Kabat AbM Chothia Contact vhCDR1 26-3527-38 31-35 26-35 26-32 30-35 vhCDR2 50-65 56-65 50-65 50-58 52-56 47-58vhCDR3  95-102 105-117  95-102  95-102  95-102  93-101 vlCDR1 24-3427-38 24-34 24-34 24-34 30-36 vlCDR2 50-56 56-65 50-56 50-56 50-56 46-55vlCDR3 89-97 105-117 89-97 89-97 89-97 89-96

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the EU numberingsystem for Fc regions (e.g, Kabat et al., supra (1991)).

The present invention provides a large number of different CDR sets. Inthis case, a “full CDR set” in the context of the anti-CD3 componentcomprises the three variable light and three variable heavy CDRs, e.g. avlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. As will beappreciated by those in the art, each set of CDRs, the VH and VL CDRs,can bind to antigens, both individually and as a set. For example, inconstrained Fv domains, the vhCDRs can bind, for example to CD3 and thevlCDRs can bind to CD3, but in the constrained format they cannot bindto CD3.

In the context of a single domain ABD (“sdABD”) such as are generallyused herein to bind to target tumor antigens (TTA), a CDR set is onlythree CDRs; these are sometimes referred to in the art as “VHH” domainsas well.

These CDRs can be part of a larger variable light or variable heavydomain, respectfully. In addition, as more fully outlined herein, thevariable heavy and variable light domains can be on separate polypeptidechains or on a single polypeptide chain in the case of scFv sequences,depending on the format and configuration of the moieties herein.

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding sites. “Epitope” refers to a determinantthat interacts with a specific antigen binding site in the variableregions known as a paratope. Epitopes are groupings of molecules such asamino acids or sugar side chains and usually have specific structuralcharacteristics, as well as specific charge characteristics. A singleantigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecific antigen binding peptide; in other words, the amino acid residueis within the footprint of the specific antigen binding peptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.” As outlined below,the invention not only includes the enumerated antigen binding domainsand antibodies herein, but those that compete for binding with theepitopes bound by the enumerated antigen binding domains.

The variable heavy and variable light domains of the invention can be“active” or “inactive”.

As used herein, “inactive VH” (“iVH”) and “inactive VL” (“iVL”) refer tocomponents of a pseudo Fv domain, which, when paired with their cognateVL or VH partners, respectively, form a resulting VH/VL pair that doesnot specifically bind to the antigen to which the “active” VH or“active” VL would bind were it bound to an analogous VL or VH, which wasnot “inactive”. Exemplary “inactive VH” and “inactive VL” domains areformed by mutation of a wild type VH or VL sequence as more fullyoutlined below. Exemplary mutations are within CDR1, CDR2 or CDR3 of VHor VL. An exemplary mutation includes placing a domain linker withinCDR2, thereby forming an “inactive VH” or “inactive VL” domain. Incontrast, an “active VH” or “active VL” is one that, upon pairing withits “active” cognate partner, i.e., VL or VH, respectively, is capableof specifically binding to its target antigen. Thus, it should beunderstood that a pseudo Fv can be a VH/iVL pair, a iVH/VL pair, or aiVH/iVL pair.

In contrast, as used herein, the term “active” refers to a CD-3 bindingdomain that is capable of specifically binding to CD-3. This term isused in two contexts: (a) when referring to a single member of an Fvbinding pair (i.e., VH or VL), which is of a sequence capable of pairingwith its cognate partner and specifically binding to CD-3; and (b) thepair of cognates (i.e., VH and VL) of a sequence capable of specificallybinding to CD-3. An exemplary “active” VH, VL or VH/VL pair is a wildtype or parent sequence.

“CD-x” refers to a duster of differentiation (CD) protein. In exemplaryembodiments, CD-x is selected from those CD proteins having a role inthe recruitment or activation of T-cells in a subject to whom apolypeptide construct of the invention has been administered. In anexemplary embodiment, CD-x is CD3, the sequence of which is shown inFIG. 5.

The term “binding domain” characterizes, in connection with the presentinvention, a domain which (specifically) binds to/interactswith/recognizes a given target epitope or a given target site on thetarget molecules (antigens), for example: EGFR and CD-3, respectively.The structure and function of the target antigen binding domain(recognizing EGFR), and preferably also the structure and/or function ofthe CD-3 binding domain (recognizing CD3), is/are based on the structureand/or function of an antibody, e.g. of a full-length or wholeimmunoglobulin molecule, including sdABDs. According to the invention,the target antigen binding domain is generally characterized by thepresence of three CDRs that bind the target tumor antigen (generallyreferred to in the art as variable heavy domains, although nocorresponding light chain CDRs are present). Alternatively, ABDs to TTAscan include three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VLregion) and/or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of theVH region). The CD-3 binding domain preferably also comprises at leastthe minimum structural requirements of an antibody which allow for thetarget binding. More preferably, the CD-3 binding domain comprises atleast three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region)and/or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VHregion). It is envisaged that in exemplary embodiments the targetantigen and/or CD-3 binding domain is produced by or obtainable byphage-display or library screening methods.

By “domain” as used herein is meant a protein sequence with a function,as outlined herein. Domains of the invention include tumor targetantigen binding domains (TTA domains), variable heavy domains, variablelight domains, linker domains, and half life extension domains.

By “domain linker” herein is meant an amino acid sequence that joins twodomains as outlined herein. Domain linkers can be cleavable linkers,constrained cleavable linkers, non-cleavable linkers, constrainednon-cleavable linkers, scFv linkers, etc.

By “cleavable linker” (“CL”) herein is meant an amino acid sequence thatcan be cleaved by a protease, preferably a human protease in a diseasetissue as outlined herein. Cleavable linkers generally are at least 3amino acids in length, with from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or more amino acids finding use in the invention, depending on therequired flexibility. A number of cleavable linker sequences are foundin FIG. 6 and FIG. 5.

By “non cleavable linker” (“NCL”) herein is meant an amino acid sequencethat cannot be cleaved by a human protease under normal physiologicalconditions.

By “constrained cleavable linker” (“CCL”) herein is meant a shortpolypeptide that contains a protease cleavage site (as defined herein)that joins two domains as outlined herein in such a manner that the twodomains cannot significantly interact with each other until after theyreside on different polypeptide chains, e.g. after cleavage. When theCCL joins a VH and a VL domain as defined herein, the VH and VL cannotself-assemble to form a functional Fv prior to cleavage due to stericconstraints in an intramolecular way (although they may assemble intopseudo Fv domains in an intermolecular way). Upon cleavage by therelevant protease, the VH and VL can assemble to form an active antigenbinding domain in an intermolecular way. In general, CCLs are less than10 amino acids in length, with 9, 8, 7, 6, 5 and 4 amino acids findinguse in the invention. In general, protease cleavage sites generally areat least 4+ amino acids in length to confer sufficient specificity, asis shown in FIG. 6.

By “constrained non-cleavable linker” (“CNCL”) herein is meant a shortpolypeptide that that joins two domains as outlined herein in such amanner that the two domains cannot significantly interact with eachother, and that is not significantly cleaved by human proteases underphysiological conditions.

By “constrained Fv domain” herein is meant an Fv domain that comprisesan active variable heavy domain and an active variable light domain,linked covalently with a constrained linker as outlined herein, in sucha way that the active heavy and light variable domains cannotintramolecularly interact to form an active Fv that will bind an antigensuch as CD3. Thus, a constrained Fv domain is one that is similar to anscFv but is not able to bind an antigen due to the presence of aconstrained linker (although they may assemble intermolecularly withinert variable domains to form pseudo Fv domains).

By “pseudo Fv domain” herein is meant a domain that comprises a pseudoor inactive variable heavy domain or a pseudo or inactive variable lightdomain, or both, linked using a domain linker (which can be cleavable,constrained, non-cleavable, non-constrained, etc.). The iVH and iVLdomains of a pseudo Fv domain do not bind to a human antigen when eitherassociated with each other (iVH/iVL) or when associated with an activeVH or VL; thus iVH/iVL, iVH/VL and iVL/VH Fv domains do not appreciablybind to a human protein, such that these domains are inert in the humanbody.

By “single chain Fv” or “scFv” herein is meant a variable heavy (VH)domain covalently attached to a variable light (VL) domain, generallyusing a domain linker as discussed herein, to form a scFv or scFvdomain. A scFv domain can be in either orientation from N- to C-terminus(VH-linker-VL or VL-linker-VH).

By “single domain Fv”, “sdFv” or “sdABD” herein is meant an antigenbinding domain that only has three CDRs, generally based on camelidantibody technology. See: Protein Engineering 9(7):1129-35 (1994); RevMol Biotech 74:277-302 (2001); Ann Rev Biochem 82:775-97 (2013). Asoutlined herein, there are two general types of sdABDs used herein:sdABDs that bind to TTAs, and are annotated as such (sdABD-TTA for thegeneric term, or sdABD-EGFR for one that binds to EGFR, sdABD-FOLR1 forone that binds to FOLR1, etc.) and sdABDs that bind to HSA (“sdABD-HSA”or “sdABD(½)”.

By “protease cleavage site” refers to the amino acid sequence recognizedand cleaved by a protease. Suitable protease cleavage sites are outlinedbelow and shown in FIG. 5 and FIG. 6.

As used herein, “protease cleavage domain” refers to the peptidesequence incorporating the “protease cleavage site” and any linkersbetween individual protease cleavage sites and between the proteasecleavage site(s) and the other functional components of the constructsof the invention (e.g., VH, VL, iVH, iVL, target antigen bindingdomain(s), half-life extension domain, etc.). As outlined herein, aprotease cleavage domain may also include additional amino acids ifnecessary, for example to confer flexibility.

The term “COBRA™” and “conditional bispecific redirected activation”refers to a bispecific conditionally effective protein that has a numberof functional protein domains. In some embodiments, one of thefunctional domains is an antigen binding domain (ABD) that binds atarget tumor antigen (TTA). In certain embodiments, another domain is anABD that binds to a T cell antigen under certain conditions. The T cellantigen includes but is not limited to CD3. The term “hemi-COBRA™”refers to a conditionally effective protein that can bind a T cellantigen when a variable heavy chain of a hemi-COBRA can associate to avariable light chain of another hemi-COBRA™ (a complementaryhemi-COBRA™) due to innate self-assembly when concentrated on thesurface of a target expressing cell.

VII. FUSION PROTEINS OF THE INVENTION

The fusion proteins of the invention have a number of differentcomponents, generally referred to herein as domains, that are linkedtogether in a variety of ways. Some of the domains are binding domains,that each bind to a target antigen (e.g. a TTA or CD3, for example). Asthey bind to more than one antigen, they are referred to herein as“multispecific”; for example, a prodrug construct of the invention maybind to a TTA and CD3, and thus are “bispecific”. A protein can alsohave higher specificities; for example, if the first aTTA binds to EGFR,the second to EpCAM and there is an anti-CD3 binding domain, this wouldbe a “trispecific” molecule. Similarly, the addition of an anti-HSAbinding domain to this construct would be “tetraspecific”, as shown inFIG. 3B.

As will be appreciated by those in the art, the proteins of theinvention can have different valencies as well as be multispecific. Thatis, proteins of the invention can bind a target with more than onebinding site; for example, Pro140 is bivalent for EGFR.

The proteins of the invention can include CD3 antigen binding domainsarranged in a variety of ways as outlined herein, tumor target antigenbinding domains, half-life extension domains, linkers, etc.

A. CD3 Antigen Binding Domains

The specificity of the response of T cells is mediated by therecognition of antigen (displayed in context of a majorhistocompatibility complex, MHC) by the T cell receptor complex. As partof the T cell receptor complex, CD3 is a protein complex that includes aCD3γ (gamma) chain, a CD3δ (delta) chain, two CD3e (epsilon) chains andtwo CD3ζ (zeta) chains, which are present at the cell surface. CD3molecules associate with the α (alpha) and β (beta) chains of the T cellreceptor (TCR) to comprise the TCR complex. Clustering of CD3 on Tcells, such as by Fv domains that bind to CD3 leads to T cell activationsimilar to the engagement of the T cell receptor but independent of itsclonal-typical specificity.

However, as is known in the art, CD3 activation can cause a number oftoxic side effects, and accordingly the present invention is directed toproviding active CD3 binding of the polypeptides of the invention onlyin the presence of tumor cells, where specific proteases are found, thatthen cleave the prodrug polypeptides of the invention to provide anactive CD3 binding domain. Thus, in the present invention, binding of ananti-CD-3 Fv domain to CD-3 is regulated by a protease cleavage domainwhich restricts binding of the CD-3 Fv domain to CD-3 only in themicroenvironment of a diseased cell or tissue with elevated levels ofproteases, for example in a tumor microenvironment as is describedherein.

Accordingly, the present invention provides two sets of VH and VLdomains, an active set (VH and VL) and an inactive set (iVH and iVL)with all four being present in the prodrug construct. The construct isformatted such that the VH and VL set cannot self-associate, but ratherassociates with an inactive partner, e.g. iVH and VL and iVL and VH asis shown herein.

1. Active Anti-CD3 Variable Heavy and Variable Light Domains

There are a number of suitable active CDR sets, and/or VH and VLdomains, that are known in the art that find use in the presentinvention. For example, the CDRs and/or VH and VL domains are derivedfrom known anti-CD-3 antibodies, such as, for example, muromonab-CD-3(OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion),SP34 or I2C, TR-66 or X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7,YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141,XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2,F101.01, UCHT-1 and WT-31.

In one embodiment, the VH and VL sequences that form an active Fv domainthat binds to human CD3 are shown in FIG. 5. As is shown herein, theseactive VH (“aVH”) and active VL (“aVL”) domains can be used in differentconfigurations and Formats 1, 2, 3 and 4.

2. Inactive Anti-CD3 Variable Heavy and Variable Light Domains

The inactive iVH and iVL domains contain “regular” framework regions(FRs) that allow association, such that an inactive variable domain willassociate with an active variable domain, rendering the pair inactive,e.g. unable to bind CD3.

As will be appreciated by those in the art, there are a number of“inactive” variable domains that find use in the invention. Basically,any variable domain with human framework regions that allowsself-assembly with another variable domain, no matter what amino acidsare in the CDR location in the variable region, can be used. Forclarity, the inactive domains are said to include CDRs, althoughtechnically the inactive variable domains do not confer bindingcapabilities.

As will be appreciated in the art, it is generally straightforward togenerate inactive VH or VL domains, and can be done in a variety ofways. In some embodiments, the generation of inactive variable domainsis generally done by altering one or more of the CDRs of an active Fv,including making changes in one or more of the three CDRs of an activevariable domain. This can be done by making one or more amino acidsubstitutions at functionally important residues in one or more CDRs,replacing some or all CDR residues with random sequences, replacing oneor more CDRs with tag or flag sequences, and/or swapping CDRs and/orvariable regions with those from an irrelevant antibody (one directed toa different organism's protein for example.

In some cases, only one of the CDRs in a variable region can be alteredto render it inactive, although other embodiments include alterations inone, two, three, four, five or six CDRs.

In some cases, the inactive domains can be engineered to promoteselective binding in the prodrug format, to encourage formation ofintramolecular iVH-VL and VH-iVL domains prior to cleavage (over, forexample, intermolecular pair formation). See for example Igawa et al.,Protein Eng. Des. Selection 23(8):667-677 (2010), hereby expresslyincorporated by reference in its entirety and specifically for theinterface residue amino acid substitutions.

In certain embodiments, the CD-3 binding domain of the polypeptideconstructs described herein exhibit not only potent CD-3 bindingaffinities with human CD-3, but show also excellent cross reactivitywith the respective cynomolgus monkey CD-3 proteins. In some instances,the CD-3 binding domain of the polypeptide constructs is cross-reactivewith CD-3 from cynomolgus monkey. In certain instances,human:cynomolgous KD ratios for CD-3 are between 5 and 0.2.

In some embodiments, the CD-3 binding domain of the antigen bindingprotein can be any domain that binds to CD-3 including but not limitedto domains from a monoclonal antibody, a polyclonal antibody, arecombinant antibody, a human antibody, a humanized antibody. In someinstances, it is beneficial for the CD-3 binding domain to be derivedfrom the same species in which the antigen binding protein willultimately be used in. For example, for use in humans, it may bebeneficial for the CD-3 binding domain of the antigen binding protein tocomprise human or humanized residues from the antigen binding domain ofan antibody or antibody fragment.

Thus, in one aspect, the antigen-binding domain comprises a humanized orhuman binding domain. In one embodiment, the humanized or humananti-CD-3 binding domain comprises one or more (e.g., all three) lightchain complementary determining region 1 (LC CDR1), light chaincomplementary determining region 2 (LC CDR2), and light chaincomplementary determining region 3 (LC CDR3) of a humanized or humananti-CD-3 binding domain described herein, and/or one or more (e.g., allthree) heavy chain complementary determining region 1 (HC CDR1), heavychain complementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) of a humanized or humananti-CD-3 binding domain described herein, e.g., a humanized or humananti-CD-3 binding domain comprising one or more, e.g., all three, LCCDRs and one or more, e.g., all three, HC CDRs.

In some embodiments, the humanized or human anti-CD-3 binding domaincomprises a humanized or human light chain variable region specific toCD-3 where the light chain variable region specific to CD-3 compriseshuman or non-human light chain CDRs in a human light chain frameworkregion. In certain instances, the light chain framework region is a λ(lambda) light chain framework. In other instances, the light chainframework region is a κ (kappa) light chain framework.

In some embodiments, one or more CD-3 binding domains are humanized orfully human. In some embodiments, one or more activated CD-3 bindingdomains have a KD binding of 1000 nM or less to CD-3 on CD-3 expressingcells. In some embodiments, one or more activated CD-3 binding domainshave a KD binding of 100 nM or less to CD-3 on CD-3 expressing cells. Insome embodiments, one or more activated CD-3 binding domains have a KDbinding of 10 nM or less to CD-3 on CD-3 expressing cells. In someembodiments, one or more CD-3 binding domains have crossreactivity withcynomolgus CD-3. In some embodiments, one or more CD-3 binding domainscomprise an amino acid sequence provided herein.

In some embodiments, the humanized or human anti-CD-3 binding domaincomprises a humanized or human heavy chain variable region specific toCD-3 where the heavy chain variable region specific to CD-3 compriseshuman or non-human heavy chain CDRs in a human heavy chain frameworkregion.

In one embodiment, the anti-CD-3 binding domain is an Fv comprising alight chain and a heavy chain of an amino acid sequence provided herein.In an embodiment, the anti-CD-3 binding domain comprises: a light chainvariable region comprising an amino acid sequence having at least one,two or three modifications (e.g., substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions) of an amino acid sequenceof a light chain variable region provided herein, or a sequence with95-99% identity with an amino acid sequence provided herein; and/or aheavy chain variable region comprising an amino acid sequence having atleast one, two or three modifications (e.g., substitutions) but not morethan 30, 20 or 10 modifications (e.g., substitutions) of an amino acidsequence of a heavy chain variable region provided herein, or a sequencewith 95-99% identity to an amino acid sequence provided herein. In oneembodiment, the humanized or human anti-CD-3 binding domain is a scFv,and a light chain variable region comprising an amino acid sequencedescribed herein, is attached to a heavy chain variable regioncomprising an amino acid sequence described herein, via a scFv linker.The light chain variable region and heavy chain variable region of ascFv can be, e.g., in any of the following orientations: light chainvariable region-scFv linker-heavy chain variable region or heavy chainvariable region-scFv linker-light chain variable region.

In some embodiments, CD-3 binding domain of an antigen binding proteinhas an affinity to CD-3 on CD-3 expressing cells with a KD of 1000 nM orless, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nMor less, 1 nM or less, or 0.5 nM or less. In some embodiments, the CD-3binding domain of an antigen binding protein has an affinity to CD-3Ewith a KD of 1000 nM or less, 100 nM or less, 50 nM or less, 20 nM orless, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. Infurther embodiments, CD-3 binding domain of an antigen binding proteinhas low affinity to CD-3, i.e., about 100 nM or greater.

The affinity to bind to CD-3 can be determined, for example, by theability of the antigen binding protein itself or its CD-3 binding domainto bind to CD-3 coated on an assay plate; displayed on a microbial cellsurface; in solution; etc., as is known in the art, generally usingBiacore or Octet assays. The binding activity of the antigen bindingprotein itself or its CD-3 binding domain of the present disclosure toCD-3 can be assayed by immobilizing the ligand (e.g., CD-3) or theantigen binding protein itself or its CD-3 binding domain, to a bead,substrate, cell, etc. Agents can be added in an appropriate buffer andthe binding partners incubated for a period of time at a giventemperature. After washes to remove unbound material, the bound proteincan be released with, for example, SDS, buffers with a high pH, and thelike and analyzed, for example, by Surface Plasmon Resonance (SPR).

In many embodiments, preferred active and inert binding domains arethose shown in FIG. 5.

B. Antigen Binding Domains to Tumor Target Antigens

In addition to the described CD3 and half-life extension domains, thepolypeptide constructs described herein also comprise target domainsthat bind to one or more target antigens or one or more regions on asingle target antigen. It is contemplated herein that a polypeptideconstruct of the invention is cleaved, for example, in adisease-specific microenvironment or in the blood of a subject at theprotease cleavage domain and that each target antigen binding domainwill bind to a target antigen on a target cell, thereby activating theCD3 binding domain to bind a T cell. In general, the TTA binding domainscan bind to their targets before protease cleavage, so they can “wait”on the target cell to be activated as T-cell engagers. At least onetarget antigen is involved in and/or associated with a disease, disorderor condition. Exemplary target antigens include those associated with aproliferative disease, a tumorous disease, an inflammatory disease, animmunological disorder, an autoimmune disease, an infectious disease, aviral disease, an allergic reaction, a parasitic reaction, agraft-versus-host disease or a host-versus-graft disease. In someembodiments, a target antigen is a tumor antigen expressed on a tumorcell. Alternatively in some embodiments, a target antigen is associatedwith a pathogen such as a virus or bacterium. At least one targetantigen may also be directed against healthy tissue.

In some embodiments, a target antigen is a cell surface molecule such asa protein, lipid or polysaccharide. In some embodiments, a targetantigen is a on a tumor cell, virally infected cell, bacteriallyinfected cell, damaged red blood cell, arterial plaque cell, or fibrotictissue cell.

Preferred embodiments of the invention utilize sdABDs as the targetingdomains. These are preferred over scFv ABDs, since the addition of otherVH and VL domains into a construct of the invention may complicate theformation of pseudo Fv domains.

In some embodiments, the pro-drug constructs of the invention utilize asingle TTA binding domain, such as generally depicted in FIG. 3A, aspairs of sdABD-TTAs, and FIG. 4, as a “format 4” configuration. FIG. 4shows the use of a single anti-EGFR ABD, although other TTA bindingdomains can be used.

In some embodiments, particularly in the Format 1 and Format 2constructs, the pro-drug constructs of the invention utilize two TTAABDs, again preferably in the sdABD-TTA format. When dual targetingdomains are used, they can bind to the same epitope of the same TTA. Forexample, as discussed herein, many of the constructs herein utilize twoidentical targeting domains. In some embodiments, two targeting domainscan be used that bind to different epitopes of the same TTA, for exampleas shown in FIG. 5, the two EGFR sdABDs bind to different epitopes onhuman EGFR. In some embodiments, the two targeting domains bind todifferent TTAs, see for example Figure.

Polypeptide constructs contemplated herein include at least one antigenbinding domain, wherein the antigen binding domain binds to at least onetarget antigen. In some embodiments, the target antigen binding domainsspecifically bind to a cell surface molecule. In some embodiments, thetarget antigen binding domains specifically bind to a tumor antigen. Insome embodiments, the target antigen binding domains specifically andindependently bind to a tumor target antigen (“TTA”) selected from atleast one of EpCAM, EGFR, HER-2, HER-3, cMet, LyPD3, B7H3, CEA, andFOLR1.

Of particular use in the present invention are sdABDs to human EGFR asshown in FIG. 5.

Additional embodiments of use in the invention are sdABDs to human FOLR1as shown in FIG. 5.

Further embodiments of use in the invention are sdABDs to human B7H3 asshown in FIG. 5.

Additional embodiments of use in the invention are sdABDs to human EpCAMas shown in FIG. 5.

In some embodiments, the protein prior to cleavage of the proteasecleavage domain is less than about 100 kDa. In some embodiments, theprotein after cleavage of the protease cleavage domain is about 25 toabout 75 kDa. In some embodiments, the protein prior to proteasecleavage has a size that is above the renal threshold for first-passclearance. In some embodiments, the protein prior to protease cleavagehas an elimination half-time of at least about 50 hours. In someembodiments, the protein prior to protease cleavage has an eliminationhall-time of at least about 100 hours. In some embodiments, the proteinhas increased tissue penetration as compared to an IgG to the sametarget antigen. In some embodiments, the protein has increased tissuedistribution as compared to an IgG to the same target antigen.

C. Half Life Extension Domains

The MCE proteins of the invention (again, also referred to herein as“COBRA™” proteins or constructs) optionally include half-life extensiondomains. Such domains are contemplated to include but are not limited toHSA binding domains, Fc domains, small molecules, and other half-lifeextension domains known in the art.

Human serum albumin (HSA) (molecular mass ˜67 kDa) is the most abundantprotein in plasma, present at about 50 mg/ml (600 uM), and has ahalf-life of around 20 days in humans. HSA serves to maintain plasma pH,contributes to colloidal blood pressure, functions as carrier of manymetabolites and fatty acids, and serves as a major drug transportprotein in plasma.

Noncovalent association with albumin extends the elimination hall-timeof short lived proteins. For example, a recombinant fusion of an albuminbinding domain to a Fab fragment resulted in a reduced in vivo clearanceof 25- and 58-fold and a half-life extension of 26- and 37-fold whenadministered intravenously to mice and rabbits respectively as comparedto the administration of the Fab fragment alone. In another example,when insulin is acylated with fatty acids to promote association withalbumin, a protracted effect was observed when injected subcutaneouslyin rabbits or pigs. Together, these studies demonstrate a linkagebetween albumin binding and prolonged action.

In one aspect, the antigen-binding proteins described herein comprise ahalf-life extension domain, for example a domain which specificallybinds to HSA. In other embodiments, the HSA binding domain is a peptide.In further embodiments, the HSA binding domain is a small molecule. Itis contemplated that the HSA binding domain of an antigen bindingprotein is fairly small and no more than 25 kD, no more than 20 kD, nomore than 15 kD, or no more than 10 kD in some embodiments. In certaininstances, the HSA binding domain is 5 kD or less if it is a peptide orsmall molecule.

In many embodiments, the half-life extension domain is a single domainantigen binding domain from a single domain antibody that binds to HSA.This domain is generally referred to herein as “sdABD” to human HSA(sdABD-HSA), or alternatively “sdABD(½)”, to distinguish these bindingdomains from the sdABDs to TTAs. A particularly useful sdABD(½) is shownin FIG. 5.

The half-life extension domain of an antigen binding protein providesfor altered pharmacodynamics and pharmacokinetics of the antigen bindingprotein itself. As above, the half-life extension domain extends theelimination half-time. The half-life extension domain also alterspharmacodynamic properties including alteration of tissue distribution,penetration, and diffusion of the antigen-binding protein. In someembodiments, the half-life extension domain provides for improved tissue(including tumor) targeting, tissue penetration, tissue distribution,diffusion within the tissue, and enhanced efficacy as compared with aprotein without a half-life extension binding domain. In one embodiment,therapeutic methods effectively and efficiently utilize a reduced amountof the antigen-binding protein, resulting in reduced side effects, suchas reduced non-tumor cell cytotoxicity.

Further, characteristics of the half-life extension domain, for examplea HSA binding domain, include the binding affinity of the HSA bindingdomain for HSA. Affinity of said HSA binding domain can be selected soas to target a specific elimination half-time in a particularpolypeptide construct. Thus, in some embodiments, the HSA binding domainhas a high binding affinity. In other embodiments, the HSA bindingdomain has a medium binding affinity. In yet other embodiments, the HSAbinding domain has a low or marginal binding affinity. Exemplary bindingaffinities include KD concentrations at 10 nM or less (high), between 10nM and 100 nM (medium), and greater than 100 nM (low). As above, bindingaffinities to HSA are determined by known methods such as SurfacePlasmon Resonance (SPR).

D. Protease Cleavage Sites

The protein compositions of the invention, and particularly the prodrugconstructs, include one or more protease cleavage sites, generallyresident in cleavable linkers, as outlined herein.

As described herein, the prodrug constructs of the invention include atleast one protease cleavage site comprising an amino acid sequence thatis cleaved by at least one protease. In some cases, the MCE proteinsdescribed herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more protease cleavage sites that are cleavedby at least one protease. As is more fully discussed herein, when morethan one protease cleavage site is used in a prodrug construction, theycan be the same (e.g. multiple sites that are cleaved by a singleprotease) or different (two or more cleavage sites are cleaved by atleast two different proteases). As will be appreciated by those in theart, constructs containing three or more protease cleavage sites canutilize one, two, three, etc.; e.g. some constructs can utilize threesites for two different proteases, etc.

The amino acid sequence of the protease cleavage site will depend on theprotease that is targeted. As is known in the art, there are a number ofhuman proteases that are found in the body and can be associated withdisease states.

Proteases are known to be secreted by some diseased cells and tissues,for example tumor or cancer cells, creating a microenvironment that isrich in proteases or a protease-rich microenvironment. In some cases,the blood of a subject is rich in proteases. In some cases, cellssurrounding the tumor secrete proteases into the tumor microenvironment.Cells surrounding the tumor secreting proteases include but are notlimited to the tumor stromal cells, myofibroblasts, blood cells, mastcells, B cells, NK cells, regulatory T cells, macrophages, cytotoxic Tlymphocytes, dendritic cells, mesenchymal stem cells, polymorphonuclearcells, and other cells. In some cases, proteases are present in theblood of a subject, for example proteases that target amino acidsequences found in microbial peptides. This feature allows for targetedtherapeutics such as antigen-binding proteins to have additionalspecificity because T cells will not be bound by the antigen bindingprotein except in the protease rich microenvironment of the targetedcells or tissue.

Proteases are proteins that cleave proteins, in some cases, in asequence-specific manner. Proteases include but are not limited toserine proteases, cysteine proteases, aspartate proteases, threonineproteases, glutamic acid proteases, metalloproteases, asparagine peptidelyases, serum proteases, Cathepsins (e.g. Cathepsin B, Cathepsin C,Cathepsin D, Cathepsin E, Cathepsin K, Cathepsin L, CathepsinS),kallikreins, hK1, hK10, hK15, KLK7, GranzymeB, plasmin, collagenase,Type IV collagenase, stromelysin, factor XA, chymotrypsin-like protease,trypsin-like protease, elastase-like protease, subtilisin-like protease,actinidain, bromelain, calpain, Caspases (e.g. Caspase-3), Mir1-CP,papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin,pepsin, matriptase, legumain, plasmepsin, nepenthesin,metalloexopeptidases, metalloendopeptidases, matrix metalloproteases(MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, meprin,urokinase plasminogen activator (uPA), enterokinase, prostate-specificantigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP(FAP-α), dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV/CD26).

Some suitable proteases and protease cleavage sequences are shown inFIG. 5 and FIG. 6.

E. Linkers

As is discussed herein, the different domains of the invention aregenerally linked together using amino acid linkers, which can conferfunctionality as well, including flexibility or inflexibility (e.g.steric constraint) as well as the ability to be cleaved using an in situprotease. These linkers can be classified in a number of ways.

The invention provides “domain linkers”, which are used to join two ormore domains (e.g. a VH and a VL, a target tumor antigen binding domain(TTABD, sometimes also referred to herein as “aTTA” (for “anti-TTA”) toa VH or VL, a half life extension domain to another component, etc.Domain linkers can be non-cleavable (NCL), cleavable (“CL”), constrainedand cleavable (CCL) and constrained and non-cleavable (CNCL), forexample.

1. Non-Cleavable Linkers

In some embodiments, the domain linker is non-cleavable. Generally,these can be one of two types: non-cleavable and flexible, allowing forthe components “upstream” and “downstream” of the linker in theconstructs to intramolecularly self-assemble in certain ways; ornon-cleavable and constrained, where the two components separated by thelinker are not able to intramolecularly self-assemble. It should benoted, however, that in the latter case, while the two component domainsthat are separated by the non-cleavable constrained linker do notintramolecularly self-assemble, other intramolecular components willself-assemble to form the pseudo Fv domains.

(i) Non-Cleavable but Flexible Linkers

In this embodiment, the linker is used to join domains to preserve thefunctionality of the domains, generally through longer, flexible domainsthat are not cleaved by in situ proteases in a patient. Examples ofinternal, non-cleavable linkers suitable for linking the domains in thepolypeptides of the invention include but are not limited to (GS)n,(GGS)n, (GGGS)n, (GGSG)n, (GGSGG)n, or (GGGGS)n, wherein n is 1, 2, 3,4, 5, 6, 7, 8, 9, or 10. In some embodiments the length of the linkercan be about 15 amino acids.

(ii) Non-Cleavable and Constrained Linkers

In some cases, the linkers do not contain a cleavage site and are alsotoo short to allow the protein domains separated by the linker tointramolecularly self-assemble, and are “constrained non-cleavablelinkers” or “CNCLs”. For example, in Pro186, an active VH and an activeVL are separated by 8 amino acids (an “8mer”) that does not allow the VHand VL to self-assemble into an active antigen binding domain. In someembodiments, the linker is still flexible; for example, (GGGS)n wheren=2. In other embodiments, although generally less preferred, more rigidlinkers can be used, such as those that include proline or bulky aminoacids.

2. Cleavable Linkers

All of the prodrug constructs herein include at least one cleavablelinker. Thus, in one embodiment, the domain linker is cleavable (CL),sometimes referred to herein as a “protease cleavage domain” (“PCD”). Inthis embodiment, the CL contains a protease cleavage site, as outlinedherein and as depicted in FIG. 5 and FIG. 6. In some cases, the CLcontains just the protease cleavage site. Optionally, depending on thelength of the cleavage recognition site, there can be an extra fewlinking amino acids at either or both of the N- or C-terminal end of theCL; for example, there may be from 1, 2, 3, 4 or 5 amino acids on eitheror both of the N- and C-termini of the cleavage site. Thus, cleavablelinkers can also be constrained (e.g. 8mers) or flexible.

Of particular interest in the present invention are MMP9 cleavablelinkers and Meprin cleavable linkers, particularly MMP9 constrainedcleavable linkers and Meprin constrained cleavable linkers.

VIII. DOMAINS OF THE INVENTION

The present invention provides a number of different formats for theprodrug polypeptides of the invention. The present invention providesconstrained Fv domains and constrained pseudo Fv domains. Additionally,the present invention provides multivalent conditionally effective(“MCE”) proteins which contain two Fv domains but are non-isomerizingconstructs. As outlined herein, these can be non-isomerizing cleavableformats or non-isomerizing non-cleavable formats, although everyconstruct contains at least one protease cleavage domain.

Importantly, while both of these domains (Fv domains and pseudo Fvdomains) are referred to herein as “constrained”, meaning that asdiscussed above and shown in FIG. 36, FIG. 37 and FIG. 38, only one ofthese needs to be constrained, although generally, when both linkers areconstrained, the protein has better expression.

Those of skill in the art will appreciate that for Formats 1, 2 and 4,there are four possibilities for the N- to C-terminal order of theconstrained and pseudo Fv domains of the invention (not showing thelinkers): aVH-aVL and iVL-iVH, aVH-aVL and iVH-iVL, aVL-aVH and iVL-iVH,aVL-aVH and iVH-iVL. All four have been tested and all four haveactivity, although the first order, aVH-aVL and iVL-iVH, shows betterexpression than the other three. Thus while the description herein isgenerally shown in this aVH-aVL and iVL-iVH format, all disclosureherein includes the other orders for these domains as well.

Note that generally, the N to C-terminal order for the full lengthconstructs of the invention is based on the aVH-aVL and iVL-iVHorientation.

Additionally, it is known in the art that there can be immunogenicity inhumans originating from the C-terminal sequences of certain ABDs.Accordingly, in general, particularly when the C-terminus of theconstructs terminates in an sdABD (for example, the sdABD-HSA domains ofmany of the constructs, a histidine tag (either His6 or His10) can beused. Many or most of the sequences herein were generated using His6C-terminal tags for purification reasons, but these sequences can alsobe used to reduce immunogenicity in humans, as is shown by Holland etal., DOI 10.1007/s10875-013-9915-0 and WO2013/024059.

A. Constrained Fv Domains

The present invention provides constrained Fv domains, that comprise anactive VH and an active VL domain that are covalently attached using aconstrained linker (which, as outlined herein, can be cleavable (Formats1 and 3) or non-cleavable (Formats 2 and 4)). The constrained linkerprevents intramolecular association between the aVH and aVL in theabsence of cleavage. Thus, a constrained Fv domain general comprises aset of six CDRs contained within variable domains, wherein the vhCDR1,vhCDR2 and vhCDR3 of the VH bind human CD-3 and the vlCDR1, vCDR2 andvlCDR3 of the VL bind human CD-3, but in the prodrug format (e.g.uncleaved), the VH and VL are unable to sterically associate to form anactive binding domain, preferring instead to pair intramolecularly withthe pseudo Fv.

The constrained Fv domains can comprise active VH and active VL (aVH andaVL) or inactive VH and VL (iVH and iVL, in which case it is aconstrained pseudo Fv domain) or combinations thereof as describedherein.

As will be appreciated by those in the art, the order of the VH and VLin a constrained Fv domain can be either (N- to C-terminal) VH-linker-VLor VL-linker-VH.

As outlined herein, for Format 1 constructs, the constrained Fv domainscan comprise a VH and a VL linked using a cleavable linker, in casessuch as those shown in FIG. 5 and FIG. 6. In this embodiment, theconstrained Fv domain has the structure (N- to C-terminus)vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4-CCL-vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4.In general, the constrained Fv domain contains active VH and VL domains(e.g. able to bind CD3 when associated) and thus has the structure (N-to C-terminus)vhFR1-avhCDR1-vhFR2-avhCDR2-vhFR3-avhCDR3-vhFR4-CCL-vlFR1-avlCDR1-vlFR2-avlCDR2-vlFR3-avlCDR3-vlFR4.

As outlined herein, for Format 2 constructs, the constrained Fv domainscan comprise a VH and a VL linked using a non-cleavable linker. In thisembodiment, the constrained Fv domain has the structure (N- toC-terminus)vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4-CNCL-vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4.In general, the constrained Fv domain contains active VH and VL domains(e.g. able to bind CD3 when associated) and thus has the structure (N-to C-terminus)vhFR1-avhCDR1-vhFR2-avhCDR2-vhFR3-avhCDR3-vhFR4-CNCL-vlFR1-avlCDR1-vlFR2-avlCDR2-vlFR3-avlCDR3-vlFR4.

Of particular use in the present invention are constrained non-cleavableFv domains having an aVH having SEQ ID NO:61, an aVL having SEQ IDNO:49, and a domain linker having SEQ ID NO:74.

B. Constrained Pseudo Fv Domains

The present invention provides constrained pseudo Fv domains, comprisinginactive or pseudo iVH and iVL domains that are covalently attachedusing a constrained linker (which, as outlined herein, can be cleavableor non-cleavable). The constrained linker prevents intramolecularassociation between the iVH and iVL in the absence of cleavage. Thus, aconstrained pseudo Fv domain general comprises an iVH and an iVL withframework regions that allow association (when in a non-constrainedformat) of the iVH and iVL, although the resulting pseudo Fv domain doesnot bind to a human protein. iVH domains can assemble with aVL domains,and iVL domains can assemble with aVH domains, although the resultingstructures do not bind to CD3.

The constrained pseudo Fv domains comprise inactive VH and VL (iVH andiVL).

As will be appreciated by those in the art, the order of the VH and VLin a constrained pseudo Fv domain can be either (N- to C-terminal)VH-linker-VL or VL-linker-VH.

As outlined herein, the constrained pseudo Fv domains can comprise a iVHand an iVL linked using a non-cleavable linker, as shown in Formats 1, 2and 4, or with cleavable linkers, as shown in Format 3.

In general, the constrained Fv domain contains inert VH and VL domains(e.g. able to bind CD3 when associated) and thus has the structure (N-to C-terminus)vhFR1-ivlCDR1-vhFR2-ivlCDR2-vhFR3-ivlCDR3-vhFR4-CNCL-vlFR1-ivhCDR1-vlFR2-ivhCDR2-vlFR3-ivhCDR3-vlFR4.

Of particular use in the present invention are constrained non-cleavablepseudo Fv domains having an iVH having SEQ ID NO:65 or SEQ ID NO:69, aniVL having SEQ ID NO:53 or SEQ ID NO:57, and a domain linker having SEQID NO:74.

IX. FORMATS OF THE INVENTION

As discussed herein, the pro-drug constructs of the invention can takeon a number of different formats, including cleavable formats with dualTTA binding domains, non-cleavable formats with dual TTA binding domains(either of which can have the same TTA binding domains or differentbinding domains), and non-cleavable formats with a single targetingdomain.

A. Cleavable Formats with Dual Targeting

The invention provides non-isomerizing cleavable formats of the “format1” type in FIG. 1. In this embodiment, the constrained Fv domaincomprise VH and VL domains that are linked using constrained cleavablelinkers and the constrained pseudo Fv domain uses constrainednon-cleavable linkers. For ease of discussion, both of these arereferred to herein as “constrained”, but as discussed above and shown inFigure, Figure and Figure, only one of these needs to be constrained,although generally, when both linkers are constrained, the protein hasbetter expression.

All constructs in Format 1 (as well as the other formats) also have acleavable linker (CL) that is cleaved by a human tumor protease.

The invention provides prodrug proteins, comprising, from N- toC-terminal, (sdABD-TTA1)-domain linker-constrained Fv domain-domainlinker-(sdABD-TTA2)-CL-constrained pseudo Fv domain-domainlinker-sdABD-HSA.

As will be appreciated by those in the art, the order of the VH and VLin either a constrained Fv domain or a constrained pseudo Fv domain canbe either (N- to C-terminal) VH-linker-VL or VL-linker-VH.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVH-CCL-iVL-domain linker-sdABD-HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVL-CCL-aVH-domainlinker-(sdABD-TTA2)-CL-iVL-CCL-iVH-domain linker-sdABD-HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVL-CCL-aVH-domainlinker-(sdABD-TTA2)-CL-iVH-CCL-iVL-domain linker-sdABD-HSA.

In some embodiments, the prodrug construct comprises sdABD(TTA1)-domainlinker-aVH-CCL-aVL-domainlinker-sdABD(TTA2)-CL-iVL-CNCL-iVH-NCL-sdABD(½). In this embodiment, theaVH, aVL, iVH and iVL have the sequences shown in FIG. 5.

In some embodiments, the prodrug construct comprises sdABD(TTA1)-domainlinker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domainlinker-sdABD(½). In this embodiment, the aVH, aVL, iVH, iVL have thesequences shown in FIG. 5. In this embodiment, the two targeting domainsbind to the same TTA, which can be EGFR, EpCAM, FOLR1 or B7H3, thesequences for which are depicted in FIG. 5.

In some embodiments, the prodrug construct comprises sdABD(TTA1)-domainlinker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domainlinker-sdABD(½). In this embodiment, the aVH, aVL, iVH, iVL have thesequences shown in FIG. 5. In this embodiment, the two targeting domainsbind to different TTAs.

In some embodiments, the prodrug construct comprises sdABD(TTA1)-domainlinker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domainlinker-sdABD(½). In this embodiment, the aVH, aVL, iVH, iVL have thesequences shown in FIG. 5. In this embodiment, the two targeting domainsbind to EGFR and EpCAM, and the sdABD-TTAs have the sequences in FIG. 5.

In some embodiments, the prodrug construct comprises sdABD(TTA1)-domainlinker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domainlinker-sdABD(½). In this embodiment, the aVH, aVL, iVH, iVL have thesequences shown in FIG. 5. In this embodiment, the two targeting domainsbind to EGFR and FOLR1, and the sdABD-TTAs have the sequences in FIG. 5.

In some embodiments, the prodrug construct comprises sdABD(TTA1)-domainlinker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domainlinker-sdABD(½). In this embodiment, the aVH, aVL, iVH, iVL have thesequences shown in FIG. 5. In this embodiment, the two targeting domainsbind to EGFR and B7H3, and the sdABD-TTAs have the sequences in FIG. 5.

In some embodiments, the prodrug construct comprises sdABD(TTA1)-domainlinker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domainlinker-sdABD(½). In this embodiment, the aVH, aVL, iVH, iVL have thesequences shown in FIG. 5. In this embodiment, the two targeting domainsbind to EpCAM and FOLR1, and the sdABD-TTAs have the sequences in FIG.5.

In some embodiments, the prodrug construct comprises sdABD(TTA1)-domainlinker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domainlinker-sdABD(½). In this embodiment, the aVH, aVL, iVH, iVL have thesequences shown in FIG. 5. In this embodiment, the two targeting domainsbind to EpCAM and B7H3, and the sdABD-TTAs have the sequences in FIG. 5.

In some embodiments, the prodrug construct comprises sdABD(TTA1)-domainlinker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domainlinker-sdABD(½). In this embodiment, the aVH, aVL, iVH, iVL have thesequences shown in FIG. 5. In this embodiment, the two targeting domainsbind to B7H3 and FOLR1, and the sdABD-TTAs have the sequences in FIG. 5.

In some embodiments, the prodrug construct comprises sdABD(TTA1)-domainlinker-aVH-CCL-aVL-domain linker-sdABD(TTA2)-CL-iVL-CNCL-iVH-domainlinker-sdABD(½). In this embodiment, the aVH, aVL, iVH, iVL have thesequences shown in FIG. 5. In this embodiment, the two targeting domainsbind to the same TTA, which can be EGFR, FOLR1, B7H3 or EpCAM, thesequences for which are depicted in FIG. 5, and the CCL and CL isselected from a linker that is cleaved by MMP9 or meprin, and thesdABD(½) has SEQ ID NO:45.

In Format 1, a preferred domain linker is SEQ ID NO:74 (which alsoserves as a preferred constrained non cleavable linker).

In Format 1, preferred constructs are Pro140 and Pro140b.

B. Non-Cleavable Formats

As shown in FIG. 2, the invention provides non-isomerizing non-cleavableformats. In this embodiment, it is understood that the “non-cleavable”applies only to the linkage of the constrained Fv domain, as there isthe activating cleavage site in the prodrug construct. In thisembodiment, the constrained Fv domain comprise VH and VL domains thatare linked using constrained non-cleavable linkers and the constrainedpseudo Fv domain uses constrained non-cleavable linkers.

As will be appreciated by those in the art, the order of the VH and VLin either a constrained Fv domain or a constrained pseudo Fv domain canbe either (N- to C-terminal) VH-linker-VL or VL-linker-VH.

The invention provides prodrug proteins, comprising, from N- toC-terminal, sdABD(TTA1)-domain linker-constrained Fv domain-domainlinker-sdABD(TTA2)-cleavable linker-constrained pseudo Fv domain-domainlinker-sdABD-HSA.

As will be appreciated by those in the art, the order of the VH and VLin either a constrained Fv domain or a constrained pseudo Fv domain canbe either (N- to C-terminal) VH-linker-VL or VL-linker-VH.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVH-CNCL-iVL-domain linker-sdABD-HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVL-CNCL-aVH-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVL-CNCL-aVH-domainlinker-(sdABD-TTA2)-CL-iVH-CNCL-iVL-domain linker-sdABD-HSA.

In some embodiments, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In thisembodiment, the aVH, aVL, iVH, iVL have the sequences shown in FIG. 5.In this embodiment, the two targeting domains bind to the same TTA,which can be EGFR, EpCAM, FOLR1 or B7H3, the sequences for which aredepicted in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In thisembodiment, the aVH, aVL, iVH, iVL have the sequences shown in FIG. 5.In this embodiment, the two targeting domains bind to different TTAs.

In some embodiments, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In thisembodiment, the aVH, aVL, iVH, iVL have the sequences shown in FIG. 5.In this embodiment, the two targeting domains bind to EGFR and EpCAM,and the sdABD-TTAs have the sequences in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In thisembodiment, the aVH, aVL, iVH, iVL have the sequences shown in FIG. 5.In this embodiment, the two targeting domains bind to EGFR and FOLR1,and the sdABD-TTAs have the sequences in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In thisembodiment, the aVH, aVL, iVH, iVL have the sequences shown in FIG. 5.In this embodiment, the two targeting domains bind to EGFR and B7H3, andthe sdABD-TTAs have the sequences in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In thisembodiment, the aVH, aVL, iVH, iVL have the sequences shown in FIG. 5.In this embodiment, the two targeting domains bind to EpCAM and FOLR1,and the sdABD-TTAs have the sequences in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In thisembodiment, the aVH, aVL, iVH, iVL have the sequences shown in FIG. 5.In this embodiment, the two targeting domains bind to EpCAM and B7H3,and the sdABD-TTAs have the sequences in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In thisembodiment, the aVH, aVL, iVH, iVL have the sequences shown in FIG. 5.In this embodiment, the two targeting domains bind to FOLR1 and B7H3,and the sdABD-TTAs have the sequences in FIG. 5.

In some embodiments, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA1)-domain linker-aVH-CNCL-aVL-domainlinker-(sdABD-TTA2)-CL-iVL-CNCL-iVH-domain linker-sdABD-HSA. In thisembodiment, the aVH, aVL, iVH, iVL have the sequences shown in FIG. 5.In this embodiment, the two targeting domains bind to the same TTA,which can be EGFR, FOLR1, B7H3 or EpCAM, the sequences for which aredepicted in FIG. 5, and the CCL and CL is selected from a linker that iscleaved by MMP9 or meprin, and the sdABD(½) has SEQ ID NO:45.

In Format 2, a preferred domain linker is SEQ ID NO:74 (which alsoserves as a preferred constrained non cleavable linker).

In Format 2, embodiments of particular use include, but are not limitedto, Pro186, Pro225, Pro226, Pro233, Pro311, Pro312, Pro313, Pro495,Pro246, Pro254, Pro255, Pro256, Pro420, Pro421, Pro432, Pro479, Pro480,Pro187, Pro221, Pro222, Pro223, Pro224, Pro393, Pro394, Pro395, Pro396,Pro429, Pro430 and Pro431.

C. Single TTA Constructs

As is shown in FIG. 4, “format 4” constructs are also included in thecompositions of the invention, that are similar to Format 2 constructsbut without a second TTA ABD. In this embodiment, it is understood thatthe “non-cleavable” applies only to the linkage of the constrained Fvdomain, as there is the activating cleavage site in the prodrugconstruct. In this embodiment, the constrained Fv domain comprise VH andVL domains that are linked using constrained non-cleavable linkers andthe constrained pseudo Fv domain uses constrained non-cleavable linkers.

As will be appreciated by those in the art, the order of the VH and VLin either a constrained Fv domain or a constrained pseudo Fv domain canbe either (N- to C-terminal) VH-linker-VL or VL-linker-VH.

The invention provides prodrug proteins, comprising, from N- toC-terminal, sdABD(TTA)-domain linker-constrained Fv domain-cleavablelinker-sdABD-HSA-constrained pseudo Fv domain. (Note that for allconstructs for this format, the sdABD-HSA does not generally have a His6tag, although it can be included).

As will be appreciated by those in the art, the order of the VH and VLin either a constrained Fv domain or a constrained pseudo Fv domain canbe either (N- to C-terminal) VH-linker-VL or VL-linker-VH.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA)-domain linker-aVH-CNCL-aVL-CL-(sdABD-HSA)-domainlinker-iVL-CNCL-iVH.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA)-domain linker-aVH-CNCL-aVL-CL-(sdABD-HSA)-domainlinker-iVH-CNCL-iVL.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA)-domain linker-aVL-CNCL-aVH-CL-(sdABD-HSA)-domainlinker-iVH-CNCL-iVL.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA)-domain linker-aVL-CNCL-aVH-CL-(sdABD-HSA)-domainlinker-iVL-CNCL-iVH.

Thus, in one embodiment, the prodrug protein comprises, from N- toC-terminal: (sdABD-TTA)-domain linker-aVH-CNCL-aVL-CL-(sdABD-HSA)-domainlinker-iVL-CNCL-iVH. In this embodiment, the aVH, aVL, iVH, iVL have thesequences shown in FIG. 5. In this embodiment, the targeting domainbinds to a TTA which can be EGFR, EpCAM, FOLR1 or B7H3, the sequencesfor which are depicted in FIG. 5.

In Format 4, a preferred domain linker is SEQ ID NO:74 (which alsoserves as a preferred constrained non cleavable linker).

In Format 4, a preferred sdABD-HSA is that of SEQ ID NO:45.

D. Two Protein Compositions

In some embodiments, the compositions of the invention comprise twodifferent molecules, sometimes referred to as “hemi-COBRAs™”, or“hemi-constructs”, that in the absence of cleavage, intramolecularlyassociate to form pseudo-Fvs. In the presence of the protease, thecleavage sites are cleaved, releasing the inert variable domains, andthe protein pair then forms an active antigen binding domain to CD3, asgenerally depicted in FIG. 3.

What is important in the design of the hemi-constructs is that theactive variable domain and the sdABD-TTA remain together after cleavage,such that the two cleaved portions are held together by the tumorantigen receptor on the tumor surface and then can form an activeanti-CD3 binding domain.

There are two different general Format 3 constructs, those wherein eachmember of the pair has a single sdABD-TTA (FIG. 3A) and those with twodifferent sdABD-TTAs, each to a different TTA (FIG. 3B).

1. Hemi-COBRA™ Constructs with Single TTA Binding Domains (Format 3A)

In some embodiments, the first hemi-COBRA™ has, from N- to C-terminal,sdABD(TTA1)-domain linker-aVH-CL-iVL-domain linker-sdABD(½) and thesecond has sdABD(½)-domain linker-iVH-CL-aVL-domain linker-sdABD(TTA2).In this embodiment, the aVH, aVL, iVH, iVL and sdABD(½) have thesequences shown in FIG. 5, and the sdABD-TTAa bind to human EGFR, EpCAM,FOLR1 and/or B7H3, and has a sequence depicted in FIG. 5.

2. Hemi-COBRA™ Constructs with Dual TTA ABDs

In some embodiments, the paired pro-drug constructs can have twosdABD-TTA binding domains per construct, as is shown in FIG. 3B. In thisembodiments, the first member of the pair comprises, from N- toC-terminal, sdABD-TTA1-domain linker-sdABD-TTA2-domainlinker-aVH-CL-iVL-domain linker-sdABD(HAS), and the second membercomprises, from N- to C-terminal, sdABD-TTA1-domainlinker-sdABD-TTA2-aVL-CL-iVH-domain linker-sdABD-HSA.

The two sdABD-TTAs on each member of the pair are different, butgenerally both members (hemi-COBRAs™) have the same two sdABD-TTAs, e.g.both have EGFR and FOLR1 or EGFR and B7H3, etc.

The two sdABD-TTAs are in some embodiments selected from the ones shownin FIG. 5.

X. METHODS OF MAKING THE COMPOSITIONS OF THE INVENTION

The pro-drug compositions of the invention are made as will generally beappreciated by those in the art and outlined below.

The invention provides nucleic acid compositions that encode thepro-drug compositions of the invention. As will be appreciated by thosein the art, the nucleic acid compositions will depend on the format ofthe pro-drug polypeptide(s). Thus, for example, when the format requirestwo amino acid sequences, such as the “format 3” constructs, two nucleicacid sequences can be incorporated into one or more expression vectorsfor expression. Similarly, prodrug constructs that are a singlepolypeptide (formats 1, 2 and 4), need a single nucleic acid in a singleexpression vector for production.

As is known in the art, the nucleic acids encoding the components of theinvention can be incorporated into expression vectors as is known in theart, and depending on the host cells used to produce the prodrugcompositions of the invention. Generally, the nucleic acids are operablylinked to any number of regulatory elements (promoters, origin ofreplication, selectable markers, ribosomal binding sites, inducers,etc.). The expression vectors can be extra-chromosomal or integratingvectors.

The nucleic acids and/or expression vectors of the invention are thentransformed into any number of different types of host cells as is wellknown in the art, including mammalian, bacterial, yeast, insect and/orfungal cells, with mammalian cells (e.g. CHO cells, 293 cells), findinguse in many embodiments.

The prodrug compositions of the invention are made by culturing hostcells comprising the expression vector(s) as is well known in the art.Once produced, traditional antibody purification steps are done,including an Protein A affinity chromatography step and/or an ionexchange chromatography step.

XI. FORMULATION AND ADMINISTRATION OF THE PRO-DRUG COMPOSITIONS OF THEINVENTION

Formulations of the pro-drug compositions used in accordance with thepresent invention are prepared for storage by mixing the pro-drugs(single proteins in the case of formats 1, 2 and 4 and two proteins inthe case of format 3) having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers (asgenerally outlined in Remington's Pharmaceutical Sciences 16th edition,Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueoussolutions.

The pro-drug compositions of the invention are administered to asubject, in accord with known methods, such as intravenousadministration as a bolus or by continuous infusion over a period oftime.

The pro-drug compositions of the invention are useful in the treatmentof cancer.

XII. EXAMPLES A. Example 1: Pro Construct Construction and Purification

Transfections

Each protein (e.g. single proteins for Formats 1, 2 and 4) or pairs ofconstructs (Format 3) were expressed from a separate expression vector(pcdna3.4 derivative). Equal amounts of plasmid DNA that encoded thepair of hemi-cobra or single chain constructs were mixed and transfectedto Expi293 cells following the manufacture's transfection protocol.Conditioned media was harvested 5 days post transfection bycentrifugation (6000 rpm×25′) and filtration (0.2 uM filter). Proteinexpression was confirmed by SDS-PAGE. Constructs were purified and thefinal buffer composition was: 25 mM Citrate, 75 mM Arginine, 75 mM NaCl,4% Sucrose, pH 7. The final preparations were stored at −80° C.

Activation of MMP9

Recombinant human (rh) MMP9 was activated according to the followingprotocol. Recombinant human MMP-9 (R&D #911-MP-010) is at 0.44 mg/ml(4.7 uM). p-aminophenylmercuric acetate (APMA) (Sigma) is prepared atthe stock concentration of 100 mM in DMSO. Assay buffer is 50 mM Tris pH7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35.

-   -   Dilute rhMMP9 with assay buffer to ˜100 ug/ml (25 ul hMMP9+75 uL        assay buffer)    -   Add p-aminophenylmercuric acetate (APMA) from 100 mM stock in        DMSO to a final concentration of 1 mM (1 uL to 100 uL)    -   Incubate at 37′C for 24 hrs    -   Dilute MMP9 to 10 ng/ul (add 900 ul of assay buffer to 100 ul of        activated solution)

The concentration of the activated rhMMP9 is ˜100 nM.

Cleavage of Constructs for TDCC Assays

To cleave the constructs, 100 ul of the protein sample at 1 mg/mlconcentration (10.5 uM) in the formulation buffer (25 mM Citric acid, 75mM L-arginine, 75 mM NaCl, 4% sucrose) was supplied with CaCl2 up to 10mM. Activated rhMMP9 was added to the concentration 20-35 nM. The samplewas incubated at room temperature overnight (16-20 hrs). Thecompleteness of cleavage was verified using SDS PAGE (10-20% TG, TGrunning buffer, 200 v, 1 hr). Samples were typically 98% cleaved.

B. Example 2: T Cell Dependent Cellular Cytotoxicity (TDCC) Assays

Firefly Luciferase transduced HT-29 cells were grown to approximately80% confluency and detached with Versene (0.48 mM EDTA in PBS-Ca-Mg).Cells were centrifuged and resuspended in TDCC media (5% HeatInactivated FBS in RPMI 1640 with HEPES, GlutaMax, Sodium Pyruvate,Non-essential amino acids, and β-mercaptoethanol). Purified human Pan-Tcells were thawed, centrifuged and resuspended in TDCC media.

A coculture of HT-29_Luc cells and T cells was added to 384-well cellculture plates. Serially diluted COBRAs were then added to the cocultureand incubated at 37° C. for 48 hours. Finally, an equal volume ofSteadyGlo luciferase assay reagent was added to the plates and incubatedfor 20 minutes. The plates were read on the Perkin Elmer Envision withan exposure time of 0.1 s/well. Total luminescence was recorded and datawere analyzed on GraphPad Prism 7.

C. Example 3: General Protocol Design of the In Vivo Adoptive T CellTransfer Efficacy Model

These protocols were used in many of the experiments of the figures.Tumor cells were implanted subcutaneous (SC) in the right flank of NSG(NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice (The Jackson Laboratory, Cat.No. 005557) and allowed to grow until an established tumor with a meanvolume of around 200 mm³ was reached. In parallel human T cells werecultured in T cell media (X-VIVO 15 [Lonza, Cat. No. 04-418Q], 5% HumanSerum, 1% Penicillin/Streptomycin, 0.01 mM 2-Mercaptoethanol) in aG-Rex100M gas permeable flask (Wilson Wolf Cat. No. 81100S) withMACSiBeads from the T Cell Activation/Expansion Kit (Miltenyi Cat. No.130-091-441) for around 10 days and supplemented with recombinant humanIL-2 protein. Tumor growth in mice and human T cell activation/expansionwere coordinated so that on Day 0 of the study mice were randomized intogroups (N=6) based on tumor size; each were then injected intravenous(IV) with 2.5×10⁶ cultured human T cells and administered the first doseof the COBRA or control molecules. Mice were dosed every 3 days for 7doses (Days 0, 3, 6, 9, 12, 15 and 18) and then followed for anadditional 2-3 weeks until tumors reached >2000 mm³ in volume or thestudy was terminated. Tumor volumes were measured every 3 days.

D. Example 4: In Vivo Activity with EGFR/MMP9 Hemi-COBRA Pair Pro77 andPro53

5×10⁶ LoVo cells or 5×10⁶ HT29 cells were implanted subcutaneous in theright flank of NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice (The JacksonLaboratory, Cat. No. 005557) and allowed to grow until tumors wereestablished. In parallel human T cells were cultured in T cell media(X-VIVO 15 [Lonza, Cat. No. 04-418Q], 5% Human Serum, 1%Penicillin/Streptomycin, 0.01 mM 2-Mercaptoethanol) in a G-Rex100M gaspermeable flask (Wilson Wolf Cat. No. 81100S) with MACSiBeads from the TCell Activation/Expansion Kit (Miltenyi Cat. No. 130-091-441) for 10days and supplemented with recombinant human IL-2 protein. Tumor growthin mice and human T cell activation/expansion were coordinated so thaton Day 0 of the study mice were randomized into groups (N=6) based ontumor size; each were then injected intravenous (IV) with 2.5×10⁶cultured human T cells and administered the first dose of the COBRA orcontrol molecules. Mice were dosed every 3 days for 7 doses (Days 0, 3,6, 9, 12, 15 and 18) and then followed until tumors reach >2000 mm³ involume or the study was terminated. Groups received 0.2 mg/kg (mpk) ofthe anti-EGFR×CD3 positive control Pro51 bispecific antibody (bsAb), 0.5mpk of the negative control anti-hen egg lysozyme (HEL)×CD3 bsAb Pro98,0.5 mpk each of the MMP9 cleavable linker containing anti-EGFRhemi-COBRA pair Pro77 and Pro53, or 0.5 mpk each of the non-cleavable(NCL) linker containing anti-EGFR hemi-COBRA pair Pro74 and Pro72. Tumorvolumes were measured every 3 days.

E. Example 5: In Vivo Activity with EGFR/MMP9 COBRA Pro140

5×10⁶ LoVo cells or 5×10⁶ HT29 cells were implanted subcutaneous in theright flank of NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice (The JacksonLaboratory, Cat. No. 005557) and allowed to grow until tumors wereestablished. In parallel human T cells are cultured in T cell media(X-VIVO 15 [Lonza, Cat. No. 04-418Q], 5% Human Serum, 1%Penicillin/Streptomycin, 0.01 mM 2-Mercaptoethanol) in a G-Rex100M gaspermeable flask (Wilson Wolf Cat. No. 81100S) with MACSiBeads from the TCell Activation/Expansion Kit (Miltenyi Cat. No. 130-091-441) for 10days and supplemented with recombinant human IL-2 protein. Tumor growthin mice and human T cell activation/expansion were coordinated so thaton Day 0 of the study mice were randomized into groups (N=6) based ontumor size; each were then injected intravenous (IV) with 2.5×10⁶cultured human T cells and administered the first dose of the COBRA orcontrol molecules. Mice were dosed every 3 days for 7 doses (Days 0, 3,6, 9, 12, 15 and 18) and then followed until tumors reach >2000 mm³ involume or the study was terminated. Groups received 0.2 mpk of theanti-EGFR×CD3 positive control Pro51 bispecific antibody (bsAb), 0.5 mpkof the negative control anti-hen egg lysozyme (HEL)×CD3 bsAb Pro98, or0.5 mpk of the MMP9 cleavable linker containing anti-EGFR COBRA Pro140.Tumor volumes were measured every 3 days.

F. Example 6: In Vivo Activity with EGFR/MMP9 COBRA Pro186

5×10⁶ HT29 cells were implanted subcutaneous in the right flank of NSG(NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice (The Jackson Laboratory, Cat.No. 005557) and allowed to grow until tumors were established. Inparallel human T cells are cultured in T cell media (X-VIVO 15 [Lonza,Cat. No. 04-418Q], 5% Human Serum, 1% Penicillin/Streptomycin, 0.01 mM2-Mercaptoethanol) in a G-Rex100M gas permeable flask (Wilson Wolf Cat.No. 81100S) with MACSiBeads from the T Cell Activation/Expansion Kit(Miltenyi Cat. No. 130-091-441) for 10 days and supplemented withrecombinant human IL-2 protein. Tumor growth in mice and human T cellactivation/expansion were coordinated so that on Day 0 of the study micewere randomized into groups (N=6) based on tumor size; each were theninjected intravenous (IV) with 2.5×10⁶ cultured human T cells andadministered the first dose of the COBRA or control molecules. Mice weredosed every 3 days for 7 doses (Days 0, 3, 6, 9, 12, 15 and 18) and thenfollowed until tumors reach >2000 mm³ in volume or the study wasterminated. Groups received 0.1 mg/kg (mpk) of the anti-EGFR×CD3positive control Pro51 bispecific antibody (bsAb), 0.3 mpk of the of thenon-cleavable (NCL) control linker containing anti-EGFR COBRA Pro214,0.1 or 0.3 mpk of the MMP9 cleavable linker containing anti-EGFR COBRAPro140, or 0.1 or 0.3 mpk of the MMP9 cleavable linker containinganti-EGFR COBRA Pro186. Tumor volumes were measured every 3 days.

G. Example: Successful Humanization of Anti-EGFR Sequences

The results are shown below.

Molecule KD (M) Kon (1/Ms) Kdis (1/s) Pro22 (parental 2.58E−09M/2.6 nM2.05E+05 5.27E−04 EGFR) Pro90 (hEGFR1) 2.00E−09M/2.0 nM 2.21E+054.40E−04 Pro48 (EGFR2) 2.89E−09M/2.9 nM 6.09E+05 1.76E−03 Pro137(hEGFR2) 4.36E−09M/4.4. nM 5.85E+05 2.55E−03 Pro51 (hEGFR2)3.27E−09M/3.2 nM 6.45E+05 2.11E−03 Pro201 (hEGFR2 2.25E−12M/2.3 pM1.55E+06 3.48E−06 with 2 binding sites)

These results show both that the humanization of the EGFR bindingdomains was successful, and that there is strong avidity to the targetEGFR when two binding sites are on the molecule.

Example: Successful Humanization of EpCAM sdABDs

The results are shown below.

Human binding Cyno binding affinity Cyno/Human cross Clone affinity (nM)(nM) reactivity VIB-13 2.3 11.6 5 hVIB-13 2.8 12.7 4.5 VIB-23 4.2 46.711.1 hVIB-23 4.1 58.8 12.6

These results show both that the humanization of the EpCAM bindingdomains was successful.

1. A protein comprising, from N- to C-terminal: a) a first single domainantigen binding domain (sdABD) that binds to a human tumor targetantigen (TTA) (sdABD-TTA); b) a first domain linker; c) a constrained Fvdomain comprising: i) a first variable heavy domain comprising a vhCDR1,vhCDR2 and vhCDR3; ii) a constrained non-cleavable linker (CNCL); andiii) a first variable light domain comprising vlCDR1, vlCDR2 and vlCDR3;d) a second domain linker; e) a second sdABD-TTA; f) a cleavable linker(CL); g) a constrained pseudo Fv domain comprising: i) a first pseudolight variable domain; ii) a non-cleavable linker (NCL); and iii) afirst pseudo heavy variable domain; h) a third domain linker; and i) athird sdABD that binds to human serum albumin; wherein said firstvariable heavy domain and said first variable light domain are capableof binding human CD3 but said constrained Fv domain does not bind CD3;wherein said first variable heavy domain and said first pseudo variablelight domain intramolecularly associate to form an inactive Fv; andwherein said first variable light domain and said first pseudo variableheavy domain intramolecularly associate to form an inactive Fv.
 2. Aprotein comprising, from N- to C-terminal: a) a first sdABD-TTA; b) afirst domain linker; c) a constrained Fv domain comprising: i) a firstvariable heavy domain comprising a vhCDR1, vhCDR2 and vhCDR3; ii) aconstrained cleavable linker (CCL); and iii) a first variable lightdomain comprising vlCDR1, vlCDR2 and vlCDR3; d) a second domain linker;e) a second sdABD-TTA; f) a cleavable linker (CL); g) a constrainedpseudo Fv domain comprising: i) a first pseudo light variable domain;ii) a non-cleavable linker (NCL); and iii) a first pseudo heavy variabledomain; h) a third domain linker; and i) a third sdABD that binds tohuman serum albumin; wherein said first variable heavy domain and saidfirst variable light domain are capable of binding human CD3 but saidconstrained Fv domain does not bind CD3; wherein said first variableheavy domain and said first pseudo variable light domainintramolecularly associate to form an inactive Fv; and wherein saidfirst variable light domain and said first pseudo variable heavy domainintramolecularly associate to form an inactive Fv.
 3. A proteinaccording to claim 1 wherein said first variable heavy domain isN-terminal to said first variable light domain and said pseudo lightvariable domain is N-terminal to said pseudo variable heavy domain.
 4. Aprotein according to claim 1 wherein said first variable heavy domain isN-terminal to said first variable light domain and said pseudo variableheavy domain is N-terminal to said pseudo variable light domain.
 5. Aprotein according to claim 1 wherein said first variable light domain isN-terminal to said first variable heavy domain and said pseudo lightvariable domain is N-terminal to said pseudo variable heavy domain.
 6. Aprotein according to claim 1 wherein said first variable light domain isN-terminal to said first variable heavy domain and said pseudo variableheavy domain is N-terminal to said pseudo variable light domain.
 7. Aprotein according to claim 1 wherein said first and second TTA is thesame.
 8. A protein according to claim 1 wherein said first and secondTTA are different.
 9. A protein according to claim 1 wherein said firstand second TTA are selected from EGFR, EpCAM, FOLR1 and B7H3.
 10. Aprotein according to claim 1 wherein said first and second sdABD-TTAsare selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, SEQID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ IDNO:29; SEQ ID NO:33; SEQ ID NO:37 and SEQ ID NO:41.
 11. A proteinaccording to claim 1 wherein said half-life extension domain has SEQ IDNO:45.
 12. A protein according to claim 1 wherein said cleavable linkeris cleaved by a human protease selected from the group consisting ofMMP2, MMP9, Meprin A, Meprin B, Cathepsin S, Cathepsin K, Cathespin L,GranzymeB, uPA, Kallekriein7, matriptase and thrombin.
 13. A proteinaccording to claim 1 comprising a protein selected from the groupconsisting of Pro186 (SEQ ID NO:145), Pro225 (SEQ ID NO:147), Pro226(SEQ ID NO:148), Pro233 (SEQ ID NO:149), Pro311 (SEQ ID NO:150), Pro312(SEQ ID NO:151), Pro313 (SEQ ID NO:152), Pro495 (SEQ ID NO:183), Pro246(SEQ ID NO:153), Pro254 (SEQ ID NO:169), Pro255 (SEQ ID NO:170), Pro256(SEQ ID NO:154), Pro420 (SEQ ID NO:155), Pro421 (SEQ ID NO:156), Pro432(SEQ ID NO:176), Pro479 (SEQ ID NO:181), Pro480 (SEQ ID NO:182), Pro187(SEQ ID NO:146), Pro221 (SEQ ID NO:165), Pro222 (SEQ ID NO:166), Pro223(SEQ ID NO:167), Pro224 (SEQ ID NO:168), Pro393 (SEQ ID NO:157), Pro394(SEQ ID NO:158), Pro395 (SEQ ID NO:159), Pro396 (SEQ ID NO:160), Pro429(SEQ ID NO:161), Pro430 (SEQ ID NO:162) and Pro431 (SEQ ID NO:163). 14.A nucleic acid encoding a protein according to claim
 1. 15. Anexpression vector comprising the nucleic acid of claim
 14. 16. A hostcell comprising the expression vector of claim
 15. 17. A method ofmaking a protein comprising culturing the host cell of claim 16 underconditions wherein said protein is expressed and recovering saidprotein.
 18. A method of treating cancer comprising administering theprotein of claim 1 to a patient.
 19. A composition comprising: a) afirst protein comprising, from N- to C-terminal: i) a first sdABD-TTA;ii) a first domain linker; iii) a pseudo Fv domain comprising, from N-to C-terminal: 1) a variable heavy chain comprising a vhCDR1, vhCDR2 andvhCDR3; 2) a cleavable linker; and 3) a first pseudo variable lightdomain comprising iVLCDR1, iVLCDR2 and iVLCDR3; iv) a second domainlinker; v) a second sdABD that binds to human serum albumin; a) a secondprotein comprising, from N- to C-terminal: i) a third sdABD that bindsto a human tumor target antigen; ii) a third domain linker; iii) apseudo Fv domain comprising, from N- to C-terminal: 1) a variable lightchain comprising a VLCDR1, VLCDR2 and VLCDR3; 2) a cleavable linker; and3) a first pseudo variable heavy domain comprising iVHCDR1, iVHCDR2 andiVHCDR3; iv) a fourth domain linker; v) a fourth sdABD that binds tohuman serum albumin; wherein said first variable heavy domain and saidfirst variable light domain are capable of binding human CD3 whenassociated; said first variable heavy domain and said first pseudovariable light domain intermolecularly associate to form an inactive Fv;said first variable light domain and said first pseudo variable heavydomain intermolecularly associate to form an inactive Fv; wherein saidfirst and third sdABD are selected from the group consisting of SEQ IDNO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ IDNO:21, SEQ ID NO:25, SEQ ID NO:29; SEQ ID NO:33; SEQ ID NO:37 and SEQ IDNO:41. 20.-27. (canceled)
 28. A composition comprising: a) a firstprotein comprising, from N- to C-terminal: i) a first sdABD-TTA; ii) afirst domain linker; iii) a second sdABD-TTA; iv) a second domainlinker; iii) a pseudo Fv domain comprising, from N- to C-terminal: 1) avariable heavy chain comprising a vhCDR1, vhCDR2 and vhCDR3; 2) acleavable linker; and 3) a first pseudo variable light domain comprisingiVLCDR1, iVLCDR2 and iVLCDR3; iv) a third domain linker; and v) asdABD-HSA; a) a second protein comprising, from N- to C-terminal: i) athird sdABD-TTA; ii) a fourth domain linker; iii) a fourth sdABD-TTA;iv) a fifth domain linker; iii) a pseudo Fv domain comprising, from N-to C-terminal: 1) a variable light chain comprising a VLCDR1, VLCDR2 andVLCDR3; 2) a cleavable linker; and 3) a first pseudo variable heavydomain comprising iVHCDR1, iVHCDR2 and iVHCDR3; iv) a sixth domainlinker; v) a sdABD-HSA; wherein said first variable heavy domain andsaid first variable light domain are capable of binding human CD3 whenassociated; said first variable heavy domain and said first pseudovariable light domain intermolecularly associate to form an inactive Fv;said first variable light domain and said first pseudo variable heavydomain intermolecularly associate to form an inactive Fv. 29.-34.(canceled)
 35. A protein comprising, from N- to C-terminal: a) a singledomain antigen binding domain (sdABD) that binds to a human tumor targetantigen (TTA) (sdABD-TTA); b) a first domain linker; c) a constrained Fvdomain comprising: i) a first variable heavy domain comprising a vhCDR1,vhCDR2 and vhCDR3; ii) a constrained non-cleavable linker (CNCL); andiii) a first variable light domain comprising vlCDR1, vlCDR2 and vlCDR3;d) a cleavable linker (CL); e) a second sdABD that binds to human serumalbumin; f) a domain linker; g) a constrained pseudo Fv domaincomprising: i) a first pseudo light variable domain; ii) a non-cleavablelinker (NCL); and iii) a first pseudo heavy variable domain; whereinsaid first variable heavy domain and said first variable light domainare capable of binding human CD3 but said constrained Fv domain does notbind CD3; said first variable heavy domain and said first pseudovariable light domain intramolecularly associate to form an inactive Fv;and said first variable light domain and said first pseudo variableheavy domain intramolecularly associate to form an inactive Fv. 36.-49.(canceled)